Methods for selectively stimulating proliferation of t cells

ABSTRACT

Methods for inducing a population of T cells to proliferate by activating the population of T cells and stimulating an accessory molecule on the surface of the T cells with a ligand which binds the accessory molecule are described. T cell proliferation occurs in the absence of exogenous growth factors or accessory cells. T cell activation is accomplished by stimulating the T cell receptor (TCR)/CD3 complex or the CD2 surface protein. To induce proliferation of an activated population T cells, an accessory molecule on the surface of the T cells, such as CD28, is stimulated with a ligand which binds the accessory molecule. The T cell population expanded by the method of the invention can be genetically transduced and used for immunotherapy or can be used in methods of diagnosis.

RELATED APPLICATIONS

[0001] This application is a continuation-in-part of the following U.S.applications: U.S. Ser. No. 08/435,816, filed May 4, 1995 entitled“Methods for Selectively Stimulating Proliferation of T cells”; U.S.Ser. No. 08/403,253, filed Mar. 10, 1995 entitled “Methods forSelectively Stimulating Proliferation of T cells”; U.S. Ser. No.08/253,964, filed Jun. 3, 1994, entitled “Methods for SelectivelyStimulating Proliferation of T cells”; U.S. Ser. No. 08/073,223, filedJun. 4, 1993, entitled “Methods for Selectively StimulatingProliferation of T cells”; U.S. Ser. No. 864,805, filed Apr. 7, 1992,entitled “CD28 Pathway Immunoregulation”; U.S. Ser. No. 864,866, filedApr. 7, 1992, entitled “Enhancement of CD28-Related Immune Response”;and U.S. Ser. No. 864,807, filed Apr. 7, 1992, entitled “ImmunotherapyInvolving Stimulation of Th CD28 Lymphokine Production”. Each of theseapplications is a continuation-in-part of U.S. Ser. No. 275,433, filedNov. 23, 1988, entitled “Immunotherapy Involving CD28 Stimulation”,which corresponds to International Application Serial No. PCT/US89/05304(Publication No. WO 90/05541) filed Nov. 22, 1989. The contents of eachof these applications is incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] The development of techniques for propagating T cell populationsin vitro has been crucial to many of the recent advances in theunderstanding of T cell recognition of antigen and T cell activation.The development of culture methods for the generation of humanantigen-specific T cell clones has been useful in defining antigensexpressed by pathogens and tumors that are recognized by T cells toestablish methods of immunotherapy to treat a variety of human diseases.Antigen-specific T cells can be expanded in vitro for use in adoptivecellular immunotherapy in which infusions of such T cells have beenshown to have anti-tumor reactivity in a tumor-bearing host. Adoptiveimmunotherapy has also been used to treat viral infections inimmunocompromised individuals.

[0003] Techniques for expanding human T cells in vitro have relied onthe use of accessory cells and exogenous growth factors, such as IL-2.The use of IL-2 and, for example, an anti-CD3 antibody to stimulate Tcell proliferation is known to expand the CD8⁺ subpopulation of T cells.The requirement for MHC-matched antigen presenting cells as accessorycells presents a significant problem for long-term culture systems.Antigen presenting cells are relatively short lived. Thus, in along-term culture system, antigen presenting cells must be continuouslyobtained from a source and replenished. The necessity for a renewablesupply of accessory cells is problematic for treatment ofimmunodeficiencies in which accessory cells are affected. In addition,when treating viral infection, accessory cells which may carry the virusmay result in contamination of the entire T cell population during longterm culture. An alternative culture method to clone and expand human Tcells in vitro in the absence of exogenous growth factor and accessorycells would be of significant benefit.

SUMMARY OF THE INVENTION

[0004] This invention pertains to methods for selectively inducing exvivo expansion of a population of T cells in the absence of exogenousgrowth factors, such as lymphokines, and accessory cells. In addition, Tcell proliferation can be induced without the need for antigen, thusproviding an expanded T cell population which is polyclonal with respectto antigen reactivity. The method provides for sustained proliferationof a selected population of CD4⁺ or CD8⁺ T cells over an extended periodof time to yield a multi-fold increase in the number of these cellsrelative to the original T cell population.

[0005] According to the method of the invention, a population of T cellsis induced to proliferate by activating the T cells and stimulating anaccessory molecule on the surface of the T cells with a ligand whichbinds the accessory molecule. Activation of a population of T cells isaccomplished by contacting the T cells with a first agent whichstimulates a TCR/CD3 complex-associated signal in the T cells.Stimulation of the TCR/CD3 complex-associated signal in a T cell isaccomplished either by ligation of the T cell receptor (TCR)/CD3 complexor the CD2 surface protein, or by directly stimulating receptor-coupledsignaling pathways. Thus, an anti-CD3 antibody, an anti-CD2 antibody, ora protein kinase C activator in conjunction with a calcium ionophore isused to activate a population of T cells.

[0006] To induce proliferation, an activated population of T cells iscontacted with a second agent which stimulates an accessory molecule onthe surface of the T cells. For example, a population of CD4⁺ T cellscan be stimulated to proliferate with an anti-CD28 antibody directed tothe CD28 molecule on the surface of the T cells. Alternatively, CD4⁺ Tcells can be stimulated with a natural ligand for CD28, such as B7-1 andB7-2. The natural ligand can be soluble, on a cell membrane, or coupledto a solid phase surface. Proliferation of a population of CD8⁺ T cellsis accomplished by use of a monoclonal antibody ES5.2D8 which binds toCD9, an accessory molecule having a molecular weight of about 27 kDpresent on activated T cells. Alternatively, proliferation of anactivated population of T cells can be induced by stimulation of one ormore intracellular signals which result from ligation of an accessorymolecule, such as CD28.

[0007] The agent providing the primary activation signal and the agentproviding the costimulatory agent can be added either in soluble form orcoupled to a solid phase surface. In a preferred embodiment, the twoagents are coupled to the same solid phase surface.

[0008] Following activation and stimulation of an accessory molecule onthe surface of the T cells, the progress of proliferation of the T cellsin response to continuing exposure to the ligand or other agent whichacts intracellularly to simulate a pathway mediated by the accessorymolecule is monitored. When the rate of T cell proliferation decreases,the T cells are reactivated and restimulated, such as with additionalanti-CD3 antibody and a co-stimulatory ligand, to induce furtherproliferation. In one embodiment, the rate of T cell proliferation ismonitored by examining cell size. Alternatively, T cell proliferation ismonitored by assaying for expression of cell surface molecules inresponse to exposure to the ligand or other agent, such as B7-1 or B7-2.The monitoring and restimulation of the T cells can be repeated forsustained proliferation to produce a population of T cells increased innumber from about 100- to about 100,000-fold over the original T cellpopulation. In a specific embodiment, a population of CD4⁺ T cells isstimulated to proliferate to produce a population of T cells increasedin number from about 10log₁₀ to 12log₁₀. In this embodiment thepopulation of CD4⁺ T cells is contacted with a solid phase surfacecomprising anti-CD3 and anti-CD28 antibodies, or a solid phase surfacecomprising anti-CD3 and a stimulatory form of B7-2. In anotherembodiment of the invention, stimulation of a population of CD28⁺ Tcells to proliferate is accompanied by selective enrichment of thepopulation in CD4⁺ T cells. The method of the invention can be used toexpand selected T cell populations for use in treating an infectiousdisease or cancer. The resulting T cell population can be geneticallytransduced and used for immunotherapy or can be used for in vitroanalysis of infectious agents such as HIV. Proliferation of a populationof CD4⁺ cells obtained from an individual infected with HIV can beachieved and the cells rendered resistant to HIV infection. The cellscan be rendered resistant to the viral infection by the addition ofantiretroviral agents to the cell culture. Alternatively, the cells canbe rendered resistant to the viral infection by culture in the presenceof an agent, such as immobilized anti-CD28 antibody, which inhibitsviral production. Following expansion of the T cell population tosufficient numbers, the expanded T cells are restored to the individual.The method of the invention also provides a renewable source of T cells.Thus, T cells from an individual can be expanded ex vivo, a portion ofthe expanded population can be readministered to the individual andanother portion can be frozen in aliquots for long term preservation,and subsequent expansion and administration to the individual.Similarly, a population of tumor-infiltrating lymphocytes can beobtained from an individual afflicted with cancer and the T cellsstimulated to proliferate to sufficient numbers and restored to theindividual.

[0009] Alternatively, the population of CD4⁺ T cells of an individual,such as an HIV infected individual, can be expanded in vivo, byadministering to the individual a biodegradable solid phase surfacecomprising a first agent that provides a primary activation signal, suchas an agent which stimulates the TCR/CD3 complex, and a second agentthat stimulates an accessory molecule on the T cell. A preferred methodof treatment of an individual having an infectious disease, such as anHIV-1 infection, consists of administering an anti-CD28 antibodyimmobilized onto a solid phase surface. The solid phase surface mayfurther comprise an agent which provides a primary activation signal. Inanother embodiment of the invention, supernatants from cultures of Tcells expanded in accordance with the method of the invention are a richsource of cytokines and can be used to sustain T cells in vivo or exvivo.

[0010] The invention also pertains to compositions comprising an agentthat provides a costimulatory signal to a T cell for T cell expansion(e.g., an anti-CD28 antibody, B7-1 or B7-2 ligand), coupled to a solidphase surface which may additionally include an agent that provides aprimary activation signal to the T cell (e.g., an anti-CD3 antibody)coupled to the same solid phase surface. These agents are preferablyattached to beads. Compositions comprising each agent coupled todifferent solid phase surfaces (i.e., an agent that provides a primary Tcell activation signal coupled to a first solid phase surface and anagent that provides a costimulatory signal coupled to a second solidphase surface) are also within the scope of this invention. Furthermore,the invention provides kits comprising the compositions, includinginstructions for use.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011]FIG. 1 depicts in vitro growth curves of CD4⁺ peripheral blood Tcells in response to culture with either an anti-CD3 antibody andinterleukin-2 (IL-2) (-), an anti-CD3 antibody and an anti-CD28antibody mAb 9.3 (⋄-⋄) or PHA only (Δ-Δ).

[0012]FIG. 2 depicts the growth curve of CD4⁺ peripheral blood T cellscultured in fetal calf serum and either anti-CD3 antibodies and IL-2(-) or an anti-CD3 antibody and an anti-CD28 antibody, mAb 9.3 (⋄-⋄).FIG. 3 depicts the growth curves of CD4⁺ peripheral blood T cellscultured in the presence of phorbol myristic acid (PMA) and ionomycinwith or without IL-2, or with an anti-CD28 antibody, mAb 9.3. Thesymbols are as follows: PMA and ionomycin (P⁺I) is represented by (□);PMA, ionomycin and IL-2 (P⁺I⁺IL-2) is represented by (); and PMA,ionomycin and anti-CD28 antibody (P⁺I⁺9.3) is represented by (♦).

[0013]FIG. 4 is a schematic representation of the selective expansion ofCD4⁺ T cells following CD28 stimulation in comparision to T cellstimulation with IL-2.

[0014]FIG. 5 depicts fluorescent activated cell sorter analysis (FACS)in which cells were stained after isolation (day 0, panel A), or after26 days in culture with either CD28 stimulation (panel B) or IL-2culture (panel C), with phycoerythrin conjugated anti-CD3, CD4, CD8 orwith an IgG2a control monoclonal antibody and fluorescence quantifiedwith a flow cytometer.

[0015]FIG. 6 shows FACS analysis of the EX5.3D10 monoclonal antibodydepicting reactivity with CD28 in comparison to an anti-CD28 monoclonalantibody 9.3. The following cell lines were tested: Panel A,untransfected CHO-DG44 cells; Panel B, CHO-HH cells; Panel C,unactivated peripheral blood lymphocytes; and Panel D, Jurkat No. 7cells.

[0016]FIG. 7 shows FACS analysis of the ES5.2D8 monoclonal antibodydepicting the binding reactivity with the following cell lines: Panel A,CHO-DG44 cells; Panel B, CHO-105A cells; Panel C, unactivated humanperipheral blood lymphocytes; and Panel D, PMA activated peripheralblood lymphocytes.

[0017]FIG. 8 is a photograph depicting immunoprecipitation analysis ofdetergent lysates of surface labeled human activated T cells indicatingthat monoclonal antibody ES5.2D8 reacts with a 27 kD cell surfaceprotein.

[0018]FIG. 9 depicts the increases in mean cell volume of CD4⁺ T cellsfollowing stimulation (S1, S2, S3, S4, S5 and S6) with an anti-CD3monoclonal antibody and an anti-CD28 monoclonal antibody over days inculture.

[0019]FIG. 10 depicts the cyclic expression of B7-1 on CD4⁺ T cellsfollowing stimulation (S1, S2, S3, S4, S5 and S6) with an anti-CD3monoclonal antibody and an anti-CD28 monoclonal antibody over days inculture.

[0020]FIG. 11 is a bar graph depicting the amount of IL-2 produced byCD4⁺ T cells following stimulation with an anti-CD3 monoclonal antibodyand an anti-CD28 monoclonal antibody or IL-2 over days in culture.

[0021]FIG. 12 is a bar graph depicting the amount ofgranulocyte-macrophage colony-stimulating factor (GM-CSF) produced byCD4⁺ T cells following stimulation with an anti-CD3 monoclonal antibodyand an anti-CD28 monoclonal antibody or IL-2 over days in culture.

[0022]FIG. 13 is a bar graph depicting the amount of tumor necrosisfactor (TNF) produced by CD4⁺ T cells following stimulation with ananti-CD3 monoclonal antibody and an anti-CD28 monoclonal antibody orIL-2 over days in culture.

[0023]FIG. 14 is a bar graph depicting the T cell receptor (TCR)diversity in CD4⁺ T cells following stimulation with an anti-CD3monoclonal antibody and an anti-CD28 monoclonal antibody at day 1 andday 24 of culture.

[0024]FIG. 15 depicts cell surface staining of CD4⁺ T cells obtainedfrom an HIV seronegative individual following stimulation (S1, S2 andS3) with an anti-CD3 monoclonal antibody and an anti-CD28 monoclonalantibody over days in culture.

[0025]FIG. 16 depicts cell surface staining of CD4⁺ T cells obtainedfrom an HIV seropositive individual following stimulation (S1, S2 andS3) with an anti-CD3 monoclonal antibody and an anti-CD28 monoclonalantibody over days in culture.

[0026]FIG. 17 depicts expansion of CD8⁺ T cells following stimulationwith an anti-CD3 monoclonal antibody and an monoclonal antibody ES5.2D8at day 4 and day 7 of culture.

[0027]FIG. 18 depicts FACS analysis with the monoclonal antibody ES5.2D8(panels C and D) or a control IgG (panels A and B) depicting the bindingreactivity with MOP cells transfected with a plasmid encoding the CD9antigen.

[0028]FIG. 19 depicts CD28⁺ T cell expansion following stimulation withanti-CD3 monoclonal antibody coated beads and anti-CD28 antibody, B7-1,B7-2, B7-1 and B7-2, or control CHO-neo cells at different time pointsafter stimulation.

[0029]FIG. 20 depicts CD28⁺ T cell expansion following stimulation withPMA and anti-CD28 antibody, B7-1, B7-2, B7-1 and B7-2, or controlCHO-neo cells at different time points after stimulation.

[0030]FIG. 21 depicts CD28⁺ T cell expansion following stimulation withanti-CD3 monoclonal antibody coated beads, B7-1, B7-2, B7-1 and B7-2, orcontrol CHO-neo cells in the presence of various amounts of antiCD28 Fabfragments and the amount of IL-2 secreted in the medium.

[0031]FIG. 22 depicts growth curves of CD4⁺ peripheral blood T cells inlong term cultures with either anti-CD3 monoclonal antibody, anti-CD28antibody, B7-1, B7-2, or control CHO-neo cells.

[0032]FIG. 23 shows the amounts of IL-2 and IL-4 produced from CD4⁺ Tcells (panels A and B), CD4⁺/CD45RA⁺ T cells (panels C and D) andCD4⁺/CD45RO⁺ T cells (panels E and F) stimulated with anti-CD3monoclonal antibody coated beads and B7-1, B7-2, or control CHO-neocells at the indicated CHO cell to T cell ratio.

[0033]FIG. 24 shows the amounts of interferon-gamma and IL-4 producedfrom CD4⁺ T cells (panels A and B), CD4⁺/CD45RA⁺ T cells (panels C andD) and CD4⁺/CD45RO⁺ T cells (panels E and F) stimulated with anti-CD3monoclonal antibody coated beads and B7-1, B7-2, or control CHO-neocells at the indicated CHO cell to T cell ratio.

[0034]FIG. 25 shows the amounts of IL-2 (panel A) and IL-4 (panel B)produced from CD4⁺ T cells stimulated with medium alone, anti-CD3monoclonal antibody and B7-1, B7-2, control CHO-neo cells after thefirst round of stimulation (day 1) or a second round of stimulation (day12).

[0035]FIG. 26 shows amounts of interferon-gamma (panel A) and IL-5(panel B) produced from CD4⁺ T cells stimulated with medium alone,anti-CD3 monoclonal antibody and B7-1, B7-2, control CHO-neo cells afterthe first round of stimulation (day 1) or a second round of stimulation(day 12).

[0036]FIG. 27 shows amounts of TNF-alpha (panel A) and GM-CSF (panel B)produced from CD4⁺ T cells stimulated with medium alone, anti-CD3monoclonal antibody and B7-1, B7-2, control CHO-neo cells after thefirst round of stimulation (day 1) or a second round of stimulation (day12).

[0037]FIG. 28 shows the amounts of IL-2 (panel A) and IL-4 (panel B)secreted from CD28⁺ T cells stimulated with medium alone, anti-CD3 andanti-CD28 antibody coated beads (“cis”), anti-CD3 and anti-CD28 antibodycoated beads and control CHO-neo cells (“cis”+CHO-neo), anti-CD3 coatedbeads and B7-1 CHO cells (anti-CD3⁺ CHO-B7-1), or anti-CD3 coated beadsand anti-CD28 coated beads (anti-CD3⁺CD28 “trans”) after initialstimulation (day 1), second stimulation (day 2), or third stimulation(day 3).

[0038]FIG. 29 depicts growth curves of the CD28⁺ T cells stimulated inthe experiment shown in FIG. 28.

[0039]FIG. 30 depicts growth curves of CD4⁺ T cells from an HIV infectedindividual stimulated with anti-CD3 and anti-CD28 antibodies in thepresence (A/D/N) or absence (No Drug) of anti-retroviral drugs.

[0040]FIG. 31 depicts growth curves of CD4⁺ T cells stimulated withanti-CD3 and anti-CD28 coated beads in the presence of added IL-2(OKT3⁺IL-2) or in the absence of added IL-2 (OKT3+9.3) in large scalecultures.

[0041]FIG. 32 depicts a growth curve of CD4⁺ T cells stimulated withanti-CD3 and anti-CD28 antibody coated beads with or without added IL-2.

[0042]FIG. 33 represent bar graphs depicting the T cell receptor (TCR)diversity in CD4⁺ T cells at day 0 (panel A) and day 71 (panel B) of aculture stimulated with anti-CD3 and anti-CD28 antibody coated beads inthe presence of added IL-2.

[0043]FIG. 34 represents growth curves of CD4 and CD8⁺ T cellsstimulated with anti-CD3 monolconal antibody (OKT3) coated beads,anti-CD3 and human B7-1Ig coated beads (OKT3/B71Ig), anti-CD3 and humanB7-2Ig coated beads (OKT3/B72Ig), or anti-CD3 and anti-CD28 monoclonalantibody 9.3 (OKT3/9.3) coated beads.

[0044]FIG. 35 represents growth curves of CD4⁺ T cells from anHIV-infected individual cultured in PHA and IL-2 (PHA+IL-2) oranti-CD3/anti-CD28 coated beads (anti-CD3⁺ anti-CD28) (Panel A); theamount of p24 in the supernatant of the cultures is shown in Panel B;the amount of gag DNA and RNA in the cells from these cultures is shownin Panels C and D, respectively.

[0045]FIG. 36 Panels A and B are graphic representations of the amountof gag RNA (Panel A) and gag DNA (Panel B) in CD4⁺ T cells fromHIV-infected patients cultured with anti-CD3/anti-CD28 antibody coatedbeads in the presence (solid symbols) or in the absence ofantiretroviral agents (open symbols).

[0046]FIG. 37 Panel A depicts a growth curve of CD4⁺ T cells from anHIV-infected individual cultured in the presence of anti-CD3/anti-CD28antibody coated beads with the antiretroviral drugs AZT, DDI, andNeviparine (A-D-N) or without antiretroviral drugs (No Drug). Panel Bdepicts the percentage of CD4⁺ T cells from an HIV-infected individualwhich are positive for CD4 and the percentage of these cells which havea specific Vβ receptor at day 0 and day 27 of culture in the presence ofanti-CD3/anti-CD28 antibody coated beads.

[0047]FIG. 38 shows the amount of IL-2, IFN-y, IL-4, IL-5, and TNF-αsecreted from CD4⁺ T cells obtained from an HIV-infected individualcultured for 10 to 20 days in the presence of anti-CD3/anti-CD28antibody coated beads.

[0048]FIG. 39, Panel A, shows the amount of p24 in the supernatant fromCD8-depleted PBMC cultured for 2 days in PHA and IL-2 (PHA/IL2), solubleanti-CD3 and anti-CD28 antibodies and IL-2 (OKT3s/9.3s/IL-2), oranti-CD3/anti-CD28 antibody coated beads (OKT3b/9.3b) and then infectedwith HIV-1_(B-AL) Panel B shows the growth curves of the cells fromwhich the level of p24 was determined in Panel A. Panel C represents theamount of p24 in the supernatant from CD8-depleted PBMC cultured in thepresence of soluble anti-CD3 and anti-CD28 antibodies (OKT3s/9.3s),soluble anti-CD3 and IL-2 (OKT3s/IL-2), soluble anti-CD3 and anti-CD-28antibodies and IL-2 (OKT3s/9.3s/IL-2), soluble anti-CD3 antibody andanti-CD28 antibody on beads (OKT3s/9.3b), and anti-CD3/anti-CD28antibodies attached to beads.

[0049]FIG. 40, Panel A, is an autoradiogram showing the amount of gagDNA in CD4⁺ T cells at 0, 2, 4, 12, 24, and 72 hours after infectionwith HIV. The cells were cultured prior to the infection and during theinfection with PHA and IL-2 (PHA+IL-2), anti-CD3 antibody coated beadsand IL-2 (CD3b+IL-2), or with anti-CD3/anti-CD28 antibody coated beads.Panel B is an autoradiogram showing the amount of a control gene,β-globin (globin). Panel C is an autoradiogram showing the hybridizationsignal obtained for 10, 100, 1,000 (1K), 10,000 (10K), and 50,000 (50K)copies of HIV-1. Panel D is an autoradiogram showing the hybridizationsignal obtained for 50, 500, 5,000 (5K), 25,000 (25K), and 50,000 (50K)cell equivalents.

[0050]FIG. 41 depicts a histogram indicating the amount of gag DNA inCD4⁺ T cells at 0, 4, 24, 72, and 120 hours after infection with HIV.The cells were cultured prior to the infection and during the infectionwith PHA and IL-2 (PHA+IL-2), PHA and anti-CD28 antibody coated beads(PHA+9.3b), or with anti-CD3/anti-CD28 antibody coated beads(OKT3b+9.3b).

DETAILED DESCRIPTION OF THE INVENTION

[0051] The methods of this invention enable the selective stimulation ofa T cell population to proliferate and expand to significant numbers invitro in the absence of exogenous growth factors or accessory cells.Interaction between the T cell receptor (TCR)/CD3 complex and antigenpresented in conjunction with either major histocompatibility complex(MHC) class I or class II molecules on an antigen-presenting cellinitiates a series of biochemical events termed antigen-specific T cellactivation. The term “T cell activation” is used herein to define astate in which a T cell response has been initiated or activated by aprimary signal, such as through the TCR/CD3 complex, but not necessarilydue to interaction with a protein antigen. A T cell is activated if ithas received a primary signaling event which initiates an immuneresponse by the T cell.

[0052] T cell activation can be accomplished by stimulating the T cellTCR/CD3 complex or via stimulation of the CD2 surface protein. Ananti-CD3 monoclonal antibody can be used to activate a population of Tcells via the TCR/CD3 complex. Although a number of anti-human CD3monoclonal antibodies are commercially available, OKT3 prepared fromhybridoma cells obtained from the American Type Culture Collection ormonoclonal antibody G19-4 is preferred. Similarly, binding of ananti-CD2 antibody will activate T cells. Stimulatory forms of anti-CD2antibodies are known and available. Stimulation through CD2 withanti-CD2 antibodies is typically accomplished using a combination of atleast two different anti-CD2 antibodies. Stimulatory combinations ofanti-CD2 antibodies which have been described include the following: theT11.3 antibody in combination with the T11.1 or T11.2 antibody (Meuer,S. C. et al. (1984) Cell 36:897-906) and the 9.6 antibody (whichrecognizes the same epitope as T11.1) in combination with the 9-1antibody (Yang, S. Y. et al. (1986) J. Immunol. 137:1097-1100). Otherantibodies which bind to the same epitopes as any of the above describedantibodies can also be used. Additional antibodies, or combinations ofantibodies, can be prepared and identified by standard techniques.

[0053] A primary activation signal can also be delivered to a T cellthrough use of a combination of a protein kinase C (PKC) activator suchas a phorbol ester (e.g., phorbol myristate acetate) and a calciumionophore (e.g., ionomycin which raises cytoplasmic calciumconcentrations). The use of these agents bypasses the TCR/CD3 complexbut delivers a stimulatory signal to T cells. These agents are alsoknown to exert a synergistic effect on T cells to promote T cellactivation and can be used in the absence of antigen to deliver aprimary activation signal to T cells.

[0054] Although stimulation of the TCR/CD3 complex or CD2 molecule isrequired for delivery of a primary activation signal in a T cell, anumber of molecules on the surface of T cells, termed accessory orcostimulatory molecules have been implicated in regulating thetransition of a resting T cell to blast transformation, and subsequentproliferation and differentiation. Thus, in addition to the primaryactivation signal provided through the TCR/CD3 complex, induction of Tcell responses requires a second, costimulatory signal. One suchcostimulatory or accessory molecule, CD28, is believed to initiate orregulate a signal transduction pathway that is distinct from thosestimulated by the TCR complex.

[0055] Accordingly, to induce an activated population of T cells toproliferate (i.e., a population of T cells that has received a primaryactivation signal) in the absence of exogenous growth factors oraccessory cells, an accessory molecule on the surface of the T cell,such as CD28, is stimulated with a ligand which binds the accessorymolecule or with an agent which acts intracellularly to stimulate asignal in the T cell mediated by binding of the accessory molecule. Inone embodiment, stimulation of the accessory molecule CD28 isaccomplished by contacting an activated population of T cells with aligand which binds CD28. Activation of the T cells with, for example, ananti-CD3 antibody and stimulation of the CD28 accessory molecule resultsin selective proliferation of CD4⁺ T cells. An anti-CD28 monoclonalantibody or fragment thereof capable of crosslinking the CD28 molecule,or a natural ligand for CD28 (e.g., a member of the B7 family ofproteins, such as B7-1(CD80) and B7-2 (CD86) (Freedman, A. S. et al.(1987) J. Immunol. 137:3260-3267; Freeman, G. J. et al. (1989) J.Immunol. 143:2714-2722; Freeman, G. J. et al. (1991) J. Exp. Med.174:625-631; Freeman, G. J. et al. (1993) Science 262:909-911; Azuma, M.et al. (1993) Nature 366:76-79; Freeman, G. J. et al. (1993) J. Exp.Med. 178:2185-2192)) can be used to induce stimulation of the CD28molecule. In addition, binding homologues of a natural ligand, whethernative or synthesized by chemical or recombinant technique, can also beused in accordance with the invention. Ligands useful for stimulating anaccessory molecule can be used in soluble form, attached to the surfaceof a cell, or immobilized on a solid phase surface as described herein.Anti-CD28 antibodies or fragments thereof useful in stimulatingproliferation of CD4⁺ T cells include monoclonal antibody 9.3, an IgG2aantibody (Dr. Jeffery Ledbetter, Bristol Myers Squibb Corporation,Seattle, Wash.), monoclonal antibody KOLT-2, an IgG1 antibody, 15E8, anIgG1 antibody, 248.23.2, an IgM antibody and EX5.3D10, an IgG2aantibody. In one specific embodiment, the molecule providing the primaryactivation signal, for example a molecule which provides stimulationthrough the TCR/CD3 complex or CD2, and the costimulatory molecule arecoupled to the same solid phase support. In particular, T cellactivation and costimulation can be provided by a solid phase surfacecontaining anti-CD3 and anti-CD28 antibodies.

[0056] A preferred anti-CD28 antibody is monoclonal antibody 9.3 orEX5.3D10. The EX5.3DI0 monoclonal antibody was derived from immunizing aBalb/c mouse with CHO (Chinese hamster ovary) cells transfected with thehuman CD28 gene (designated CHO-hh). Hybridomas from the fusion wereselected by whole cell ELISA screening against Jurkat (human T leukemia)CD28 tranfectants designated Jurkat #7. Reactivity of the EX5.3D10 withCD28 was further confirmed by fluorescent activated cell sorter analysis(FACS) analysis in which it was tested side by side with the monoclonal9.3 (FIG. 6). Neither antibody bound to untransfected CHO-DG44 cells andtheir binding profiles were nearly identical for the two CD28transfectant lines, CHO-hh and Jurkat #7, as well as normal humanperipheral blood lymphocytes. A hybridoma which produces the monoclonalantibody EX5.3D 10 has been deposited with the American Type CultureCollection on Jun. 4, 1993, at ATCC Deposit No. HB11373.

[0057] In a specific embodiment of the invention, activated T cells arecontacted with a stimulatory form of a natural ligand for CD28 forcostimulation. The natural ligands of CD28 include the members of the B7family of proteins, such as B7-1 (CD80) (SEQ ID NO:1 and 2) and B7-2(CD86) (SEQ ID NO:3 and 4). B7-1 and B7-2 are collectively referred toherein as “B7 molecules”. A “stimulatory form of a natural ligand forCD28” is a form of a natural ligand that is able to bind to CD28 andcostimulate the T cell. Costimulation can be evidenced by proliferationand/or cytokine production by T cells that have received a primaryactivation signal, such as stimulation through the CD3/TCR complex orthrough CD2.

[0058] Expression or Coupling of B7 Molecules on the Surface of Cells

[0059] In a preferred embodiment of the invention, a B7 molecule or aportion of a B7 molecule or a modified form of a B7 molecule capable ofinducing costimulation is localized on the surface of a cell. This canbe accomplished by transfecting a cell with a nucleic acid encoding theB7 molecule (e.g. B7-1, B7-2) in a form suitable for its expression onthe cell surface or alternatively by coupling a B7 molecule to the cellsurface.

[0060] The B7 molecules are preferably expressed on the surface of acell by transfection of the cell with a nucleic acid encoding the B7molecule in a form suitable for expression of the molecule on thesurface of the cell. The terms “transfection” or “transfected with”refers to the introduction of exogenous nucleic acid into a mammaliancell and encompass a variety of techniques useful for introduction ofnucleic acids into mammalian cells including electroporation,calcium-phosphate precipitation, DEAE-dextran treatment, lipofection,microinjection and infection with viral vectors. Suitable methods fortransfecting mammalian cells can be found in Sambrook et al. (MolecularCloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratorypress (1989)) and other laboratory textbooks. The nucleic acid to beintroduced may be, for example, DNA encompassing the gene(s) encodingB7-1 and/or B7-2, sense strand RNA encoding B7-1 and/or B7-2 or arecombinant expression vector containing a cDNA encoding B7-1 and/orB7-2. The nucleotide sequence of a cDNA encoding human B7-1 is shown inSEQ ID NO: 1, and the amino acid sequence of a human B7-1 protein isshown in SEQ ID NO:2. The nucleotide sequence of a cDNA encoding humanB7-2 is shown in SEQ ID NO: 3, and the amino acid sequence of a humanB7-2 protein is shown in SEQ ID NO:4. The nucleic acids encoding B7-1and B7-2 are further described in Freedman, A. S. et al. (1987) J.Immunol. 137:3260-3267; Freeman, G. J. et al. (1989) J. Immunol.143:2714-2722; Freeman, G. J. et al. (1991) J. Exp. Med. 174:625-631;Freeman, G. J. et al. (1993) Science 262:909-911; Azuma, M. et al.(1993) Nature 366:76-79 and; Freeman, G. J. et al. (1993) J. Exp. Med.178:2185-2192.

[0061] The nucleic acid is in a form suitable for expression of the B7molecule in which the nucleic acid contains all of the coding andregulatory sequences required for transcription and translation of agene, which may include promoters, enhancers and polyadenylationsignals, and sequences necessary for transport of the molecule to thesurface of the tumor cell, including N-terminal signal sequences. Whenthe nucleic acid is a cDNA in a recombinant expression vector, theregulatory functions responsible for transcription and/or translation ofthe cDNA are often provided by viral sequences. Examples of commonlyused viral promoters include those derived from polyoma, Adenovirus 2,cytomegalovirus and Simian Virus 40, and retroviral LTRs. Regulatorysequences linked to the cDNA can be selected to provide constitutive orinducible transcription, by, for example, use of an inducible promoter,such as the metallothionin promoter or a glucocorticoid-responsivepromoter. Expression of B7-1 or B7-2 on the surface of a cell can beaccomplished, for example, by including the native transmembrane codingsequence of the molecule in the nucleic acid sequence, or by includingsignals which lead to modification of the protein, such as a C-terminalinositol-phosphate linkage, that allows for association of the moleculewith the outer surface of the cell membrane.

[0062] The B7 molecule can be expressed on a cell using a plasmidexpression vector which contains nucleic acid, e.g., a cDNA, encodingthe B7 molecule. Suitable plasmid expression vectors include CDM8 (Seed,B., Nature 329, 840 (1987)) and pMT2PC (Kaufman, et al., EMBO J. 6,187-195 (1987)). Since only a small fraction of cells (about 1 out of10⁵) typically integrate transfected plasmid DNA into their genomes, itis advantageous to transfect a nucleic acid encoding a selectable markerinto the tumor cell along with the nucleic acid(s) of interest.Preferred selectable markers include those which confer resistance todrugs such as G418, hygromycin and methotrexate. Selectable markers maybe introduced on the same plasmid as the gene(s) of interest or may beintroduced on a separate plasmid. Following selection of transfectedcells using the appropriate selectable marker(s), expression of thecostimulatory molecule on the surface of the cell can be confirmed byimmunofluorescent staining of the cells. For example, cells may bestained with a fluorescently labeled monoclonal antibody reactiveagainst the costimulatory molecule or with a fluorescently labeledsoluble receptor which binds the costimulatory molecule such as CTLA4Ig.Expression of the B7 costimulatory molecule can be determined using amonoclonal antibody, such as BB1 or 133, which recognizes B7-1 or themonoclonal antibody IT2 which recognizes B7-2. Alternatively, a labeledsoluble CD28 or CTLA4 protein or fusion protein (e.g., CTLA4Ig) whichbinds to the B7 molecules can be used to detect expression of B7 on thecell surface.

[0063] The cell to be transfected can be any eukaryotic cell, preferablycells that allow high level expression of the transfected gene, such aschinese hamster ovary (CHO) cells or COS cells. The cell is mostpreferably a CHO cell and a specific protocol for transfection of thesecells is provided in Example 11.

[0064] In another embodiment, B7 molecules (e.g., B7-1, B7-2) arecoupled to the cell surface by any of a variety of different methods. Inthis embodiment, the B7 molecule to be coupled to the cell surface canbe obtained using standard recombinant DNA technology and expressionsystems which allow for production and isolation of the costimulatorymolecule(s) or obtained from a cell expressing the costimulatorymolecule, as described below for the preparation of a soluble form ofthe B7 molecules. The isolated costimulatory molecule is then coupled tothe cell. The terms “coupled” or “coupling” refer to a chemical,enzymatic or other means (e.g., antibody) by which the B7 molecule islinked to a cell such that the costimulatory molecule is present on thesurface of the cell and is capable of triggering a costimulatory signalin T cells. For example, the B7 molecule can be chemically crosslinkedto the cell surface using commercially available crosslinking reagents(Pierce, Rockford Ill.). Another approach to coupling a B7 molecule to acell is to use a bispecific antibody which binds both the costimulatorymolecule and a cell-surface molecule on the cell. Fragments, mutants orvariants of a B7 molecule which retain the ability to trigger acostimulatory signal in T cells when coupled to the surface of a cellcan also be used.

[0065] The level of B7 molecules expressed on or coupled to the cellsurface can be determined by FACS analysis, as described in Example 11.

[0066] For T cell costimulation, the B7-expressing cells can be culturedto a high density, mitomycin C treated (e.g., at 25 μg/ml for an hour),extensively washed, and incubated with the T cells to be costimulated.The ratio of T cells to B7-expressing cells can be anywhere between 10:1to 1:1, preferably 2.5:1 T cells to B7-expressing cells.

[0067] Soluble Forms of B7 Molecules as Costimulator

[0068] The natural ligands of CD28 can also be presented to T cells in asoluble form. Soluble forms of B7 molecules include natural B7 molecules(e.g., B7-1, B7-2), a fragment thereof, or modified form of the fulllength or fragment of the B7 molecule that is able to bind to CD28 andcostimulate the T cell. Costimulation can be evidenced by proliferationand/or cyotkine production by T cells that have received a primaryactivation signal. Modifications of B7 molecules include modificationsthat preferably enhance the affinity of binding of B7 molecules to CD28molecules, but also modifications that diminish or do not affect theaffinity of binding of B7 molecules to CD28 molecules. Modifications ofB7 molecules also include those that increase the stability of a solubleform of a B7 molecule. The modifications of B7 molecules are usuallyproduced by amino acid substitutions, but can also be produced bylinkage to another molecule.

[0069] In one specific embodiment, the soluble form of a B7 molecule isa fusion protein containing a first peptide consisting of a B7 molecule(e.g., B7-1, B7-2), or fragment thereof and a second peptidecorresponding to a moiety that alters the solubility, binding, affinity,stability, or valency (i.e., the number of binding sites available permolecule) of the first peptide. Preferably, the first peptide includesan extracellular domain portion of a B7 molecule (e.g., about amino acidresidues 24-245 of the B7-2 molecule having an amino acid sequence shownin SEQ ID NO: 4) that interacts with CD28 and is able to provide acostimulatory signal as evidenced by stimulation of proliferation of Tcells or secretion of cytokines from the T cells upon exposure to theB71g fusion protein and a primary T cell activation signal. Thus, aB7-1Ig fusion protein will comprise at least about amino acids 1-208(SEQ ID NO:2) of B7-1 and a B7-2Ig fusion protein will comprise at leastabout amino acids 24-245 (SEQ ID NO:4) of B7-2.

[0070] The second peptide is a fragment of an Ig molecule, such as an Fcfragment that comprises the hinge, CH2 and CH3 regions of human IgGI orIgG4. Several Ig fusion proteins have been previously described (seee.g., Capon, D. J. et al. (1989) Nature 337:525-531 and Capon U.S. Pat.No. 5,116,964 [CD4-IgGl constructs]; Linsley, P. S. et al. (1991) J.Exp. Med. 173:721-730 [a CD28-IgG1 construct and a B7-1-IgGI construct];and Linsley, P. S. et al. (1991) J. Exp. Med. 174:561-569 [aCTLA4-IgGl]). A resulting B71g fusion protein (e.g., B7-1Ig, B7-21g) mayhave altered B7-2 solubility, binding affinity, stability, or valencyand may increase the efficiency of protein purification. In particularfusion of a B7 molecule or portion thereof to the Fc region of animmunoglobulin molecule generally provides an increased stability to theprotein, in particular in the plasma.

[0071] Fusion proteins within the scope of the invention can be preparedby expression of a nucleic acid encoding the fusion protein in a varietyof different systems. Typically, the nucleic acid encoding a B7 fusionprotein comprises a first nucleotide sequence encoding a first peptideconsisting of a B7 molecule or a fragment thereof and a secondnucleotide sequence encoding a second peptide corresponding to a moietythat alters the solubility, binding, stability, or valency of the firstpeptide, such as an immunoglobulin constant region. Nucleic acidencoding a peptide comprising an immunoglobulin constant region can beobtained from human immunoglobulin mRNA present in B lymphocytes. It isalso possible to obtain nucleic acid encoding an immunoglobulin constantregion from B cell genomic DNA. For example, DNA encoding Cγ1 or Cγ4 canbe cloned from either a cDNA or a genomic library or by polymerase chainreaction (PCR) amplification in accordance standard protocols. Apreferred nucleic acid encoding an immunoglobulin constant regioncomprises all or a portion of the following: the DNA encoding human Cγ1(Takahashi, N. S. et al. (1982) Cell 29:671-679), the DNA encoding humanCγ2; the DNA encoding human Cγ3(Huck, S., et al. (1986) Nucl. Acid Res.14:1779); and the DNA encoding human Cγ4. When an immunoglobulinconstant region is used in the B7 fusion protein, the constant regioncan be modified to reduce at least one constant region mediatedbiological effector function. For example, DNA encoding a Cγ1 or Cγ4constant region can be modified by PCR mutagenesis or site directedmutagenesis. Protocols and reagents for site directed mutagenesissystems can be obtained commercially from Amersham International PLC,Amersham, UK.

[0072] In a particularly prefered embodiment of the invention, B7-1Igand B7-2Ig fusion proteins comprise about amino acids 1-208 of B7-1 (SEQID NO: 2) and about amino acids 24-245 of B7-2 (SEQ ID NO: 4),respectively, fused to the heavy chain of IgG1.

[0073] In one embodiment the first and second nucleotide sequences arelinked (i.e., in a 5′ to 3′ orientation by phosphodiester bonds) suchthat the translational frame of the B7 protein or fragment thereof andthe IgC (i.e., Fc fragment that comprises the hinge, CH2, and CH3regions of human IgG) coding segments are maintained (i.e., thenucleotide sequences are joined together in-frame). Thus, expression(i.e., transcription and translation) of the nucleotide sequenceproduces a functional B7Ig fusion protein. The nucleic acids of theinvention can be prepared by standard recombinant DNA techniques. Forexample, a B7Ig fusion protein can be constructed using separatetemplate DNAs encoding B7 and an immunoglobulin constant region. Theappropriate segments of each template DNA can be amplified by polymerasechain reaction (PCR) and ligated in frame using standard techniques. Anucleic acid of the invention can also be chemically synthesized usingstandard techniques. Various methods of chemically synthesizingpolydeoxynucleotides are known, including solid-phase synthesis whichhas been automated in commercially available DNA synthesizers (See e.g.,Itakura et al. U.S. Pat. No. 4,598,049; Caruthers et al. U.S. Pat. No.4,458,066; and Itakura U.S. Pat. Nos. 4,401,796 and 4,373,071,incorporated by reference herein).

[0074] The nucleic acids encoding B7 molecules or B7Ig fusion proteins(e.g., B7-1, B7-2) can be inserted into various expression vectors,which in turn direct the synthesis of the corresponding protein in avariety of hosts, particularly eucaryotic cells, such as mammalian orinsect cell culture and procaryotic cells, such as E. coli. Expressionvectors within the scope of the invention comprise a nucleic acid asdescribed herein and a promotor operably linked to the nucleic acid.Such expression vectors can be used to transfect host cells to therebyproduce fusion proteins encoded by nucleic acids as described herein. Anexpression vector of the invention, as described herein, typicallyincludes nucleotide sequences encoding a B7 molecule or B7Ig fusionprotein operably linked to at least one regulatory sequence. “Operablylinked” is intended to mean that the nucleotide sequence is linked to aregulatory sequence in a manner which allows expression of thenucleotide sequence in a host cell (or by a cell extract). Regulatorysequences are art-recognized and can be selected to direct expression ofthe desired protein in an appropriate host cell. The term regulatorysequence is intended to include promoters, enhancers, polyadenylationsignals and other expression control elements. Such regulatory sequencesare known to those skilled in the art and are described in Goeddel, GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990). It should be understood that the design of theexpression vector may depend on such factors as the choice of the hostcell to be transfected and/or the type and/or amount of protein desiredto be expressed.

[0075] An expression vector of the invention can be used to transfectcells, either procaryotic or eucaryotic (e.g., mammalian, insect oryeast cells) to thereby produce fusion proteins encoded by nucleotidesequences of the vector. Expression in procaryotes is most often carriedout in E. coli with vectors containing constitutive or induciblepromotors. Certain E. coli expression vectors (so called fusion-vectors)are designed to add a number of amino acid residues to the expressedrecombinant protein, usually to the amino terminus of the expressedprotein. Such fusion vectors typically serve three purposes: 1) toincrease expression of recombinant protein; 2) to increase thesolubility of the target recombinant protein; and 3) to aid in thepurification of the target recombinant protein by acting as a ligand inaffinity purification. Examples of fusion expression vectors includepGEX (Amrad Corp., Melbourne, Australia) and pMAL (New England Biolabs,Beverly, Mass.) which fuse glutathione S-tranferase and maltose Ebinding protein, respectively, to the target recombinant protein.Accordingly, a B7 molecule or B7Ig fusion gene may be linked toadditional coding sequences in a procaryotic fusion vector to aid in theexpression, solubility or purification of the fusion protein. Often, infusion expression vectors, a proteolytic cleavage site is introduced atthe junction of the fusion moiety and the target recombinant protein toenable separation of the target recombinant protein from the fusionmoiety subsequent to purification of the fusion protein. Such enzymes,and their cognate recognition sequences, include Factor Xa, thrombin andenterokinase.

[0076] Inducible non-fusion expression vectors include pTrc (Amann etal., (1988) Gene 69:301-315) and pET 11d (Studier et al., GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) 60-89). Target gene expression from the pTrcvector4 relies on host RNA polymerase transcription from the hybridtrp-lac fusion promoter. Target gene expression from the pET 11d vectorrelies on transcription from the T7 gn10-lac 0 fusion promoter mediatedby a coexpressed viral RNA polymerase (T7 gn1). This viral polymerase issupplied by host strains BL21(DE3) or HMS174(DE3) from a resident λprophage harboring a T7 gn1 under the transcriptional control of thelacUV 5 promoter. One strategy to maximize expression of at B7 moleculeor B7Ig fusion protein in E. coli is to express the protein in a hostbacteria with an impaired capacity to proteolytically cleave therecombinant protein (Gottesman, S., Gene Expression Technology: Methodsin Enzymology 185, Academic Press, San Diego, Calif. (1990) 119-128).Another strategy would be to alter the nucleotide sequence of the B7molecule or B7Ig fusion protein construct to be inserted into anexpression vector so that the individual codons for each amino acidwould be those preferentially utilized in highly expressed E. coliproteins (Wada et al. (1992) Nuc. Acids Res. 20:2111-2118). Suchalteration of nucleic acid sequences are encompassed by the inventionand can be carried out by standard DNA synthesis techniques.

[0077] Alternatively, a B7 molecule or B7Ig fusion protein can beexpressed in a eucaryotic host cell, such as mammalian cells (e.g.,Chinese hamster ovary cells (CHO) or NS0 cells), insect cells (e.g.,using a baculovirus vector) or yeast cells. Other suitable host cellsmay be found in Goeddel, (1990) supra or are known to those skilled inthe art. Eucaryotic, rather than procaryotic, expression of a B7molecule or B7Ig may be preferable since expression of eucaryoticproteins in eucaryotic cells can lead to partial or completeglycosylation and/or formation of relevant inter- or intra-chaindisulfide bonds of a recombinant protein. For expression in mammaliancells, the expression vector's control functions are often provided byviral material. For example, commonly used promoters are derived frompolyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. To express aB7 molecule or B7Ig fusion protein in mammalian cells, generally COScells (Gluzman, Y., (1981) Cell 23:175-182) are used in conjunction withsuch vectors as pCDM8 (Seed, B., (1987) Nature 329:840) for transientamplification/expression, while CHO (dhfr⁻ Chinese Hamster Ovary) cellsare used with vectors such as pMT2PC (Kaufman et al. (1987), EMBO J.6:187-195) for stable amplification/expression in mammalian cells. Apreferred cell line for production of recombinant protein is the NSOmyeloma cell line available from the ECACC (catalog #85110503) anddescribed in Galfre, G. and Milstein, C. ((1981) Methods in Enzymology73(13):3-46; and Preparation of Monoclonal Antibodies: Strategies andProcedures, Academic Press, N.Y., N.Y). Examples of vectors suitable forexpression of recombinant proteins in yeast (e.g., S. cerivisae) includepYepSec1 (Baldari.et al., (1987) Embo J. 6:229-234), pMFa (Kurjan andHerskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al, (1987) Gene54:113-123), and pYES2 (Invitrogen Corporation, San Diego, Calif.).Baculovirus vectors available for expression of proteins in culturedinsect cells (SF 9 cells) include the pAc series (Smith et al., (1983)Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow, V. A., andSummers, M. D., (1989) Virology 170:31-39).

[0078] Vector DNA can be introduced into procaryotic or eucaryotic cellsvia conventional transformation or transfection techniques such ascalcium phosphate or calcium choloride co-precipitation,DEAE-dextran-mediated transfection, lipofection, or electroporation.Suitable methods for transforming host cells can be found in Sambrook etal. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold SpringHarbor Laboratory press (1989)), and other laboratory textbooks.

[0079] For stable transfection of mammalian cells, it is known that,depending upon the expression vector and transfection technique used,only a small faction of cells may integrate DNA into their genomes. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those which confer resistance todrugs, such as G418, hygromycin and methotrexate. Nucleic acid encodinga selectable marker may be introduced into a host cell on the sameplasmid as the gene of interest or may be introduced on a separateplasmid. Cells containing the gene of interest can be identified by drugselection (e.g., cells that have incorporated the selectable marker genewill survive, while the other cells die). The surviving cells can thenbe screened for production of B7 molecules or B7Ig fusion proteins by,for example, immunoprecipitation from cell supernatant with an anti-B7monoclonal antibody.

[0080] B7 molecules or B7 Ig fusion proteins produced by recombinanttechnique may be secreted and isolated from a mixture of cells andmedium containing the protein. Alternatively, the protein may beretained cytoplasmically and the cells harvested, lysed and the proteinisolated. A cell culture typically includes host cells, media and otherbyproducts. Suitable mediums for cell culture are well known in the art.Protein can be isolated from cell culture medium, host cells, or bothusing techniques known in the art for purifying proteins.

[0081] For T cell costimulation, the soluble forms of the naturalligands for CD28 are added to the T cell culture in an amount sufficientto result in costimulation of activated T cells. The appropriate amountof soluble ligand to be added will vary with the specific ligand, butcan be determined by assaying different amounts of the soluble ligand inT cell cultures and measuring the extent of costimulation byproliferation assays or production of cytokines, as described in theExamples.

[0082] Coupling of the Natural Ligands to a Solid Phase Surface

[0083] In another embodiment of the invention, a natural ligand of CD28(B7-1, B7-2) can be presented to T cells in a form attached to a solidphase surface, such as beads. The B7 molecules, fragments thereof ormodified forms thereof capable of binding to CD28 and costimulating theT cells (e.g., B7 fusion proteins) can be prepared as described for thesoluble B7 forms. These molecules can then be attached to the solidphase surface via several methods. For example the B7 molecules can becrosslinked to the beads via covalent modification using tosyl linkage.In this method, B7 molecules or B7 fusion proteins are in 0.,05M boratebuffer, pH 9.5 and added to tosyl activated magnetic immunobeads (DynalInc., Great Neck, N.Y.) according to manufacturer's instructions. Aftera 24 hr incubation at 22° C., the beads are collected and washedextensively. It is not mandatory that immunmagnetic beads be used, asother methods are also satisfactory. For example, the B7 molecules mayalso be immobilized on polystyrene beads or culture vessel surfaces.Covalent binding of the B7 molecules or B7Ig fusion proteins to thesolid phase surface is preferrable to adsorption or capture by asecondary monoclonal antibody. B7Ig fusion proteins can be attached tothe solid phase surface through anti-human IgG molecules bound to thesolid phase surface. In particular, beads to which anti-human IgGmolecules are bound can be obtained from Advanced Magnetics, Inc. Thesebeads can then be incubated with the B7Ig fusion proteins in anappropriate buffer such as PBS for about an hour at 5° C., and theuncoupled B7Ig proteins removed by washing the beads in a buffer, suchas PBS.

[0084] It is also possible to attach the B7 molecules to the solid phasesurface through an avidin- or streptavidin-biotin complex. In thisparticular embodiment, the soluble B7 molecule is first crosslinked tobiotin and then reacted with the solid phase surface to which avidin orstreptavidin molecules are bound. It is also possible to crosslink theB7 molecules with avidin or streptavidin and to react these with a solidphase surface that is covered with biotin molecules.

[0085] The amount of B7 molecules attached to the solid phase surfacecan be determined by FACS analysis if the solid phase surface is that ofbeads or by ELISA if the solid phase surface is that of a tissue culturedish. Antibodies reactive with the B7 molecules, such as mAb BBI, mAbIT2, and mAb 133 can be used in these assays. Alternatively, CTLA4Ig canalso be used for that purpose.

[0086] In a specific embodiment, the stimulatory form of a B7 moleculeis attached to the same solid phase surface as the agent that stimulatesthe TCR/CD3 complex, such as an anti-CD3 antibody. In addition toanti-CD3, other antibodies that bind to receptors that mimic antigensignals may be used, for example, the beads or other solid phase surfacemay be coated with combinations of anti-CD2 and a B7 molecule. The twostimulatory molecules can be bound to the solid phase surface in variousratios, but preferably in equimolar amounts.

[0087] In a typical experiment, B7-coated beads or beads coated with B7molecules and an agent that stimulates the TCR/CD3 complex will be addedat a ratio of 3 beads per T cell.

[0088] Agents which Act Intracellularly to Stimulate a Signal Associatedwith CD28 Ligation

[0089] In another embodiment of the invention, an activated populationof CD4⁺ T cells is stimulated to proliferate by contacting the T cellswith an agent which acts intracellularly to stimulate a signal in the Tcell mediated by ligation of an accessory molecule, such as CD28. Theterm “agent”, as used herein, is intended to encompass chemicals andother pharmaceutical compounds which stimulate a costimulatory or othersignal in a T cell without the requirement for an interaction between aT cell surface receptor and a costimulatory molecule or other ligand.For example, the agent may act intracellularly to stimulate a signalassociated with CD28 ligation. In one embodiment, the agent is anon-proteinaceous compound. As the agent used in the method is intendedto bypass the natural receptor:ligand stimulatory mechanism, the termagent is not intended to include a cell expressing a natural ligand.Natural ligands for CD28 include members of the B7 family of proteins,such as B7-1(CD80) and B7-2 (CD86).

[0090] It is known that CD28 receptor stimulation leads to theproduction of D-3 phosphoinositides in T cells and that inhibition ofthe activity of phosphatidylinositol 3-kinase (PI3K) in a T cell caninhibit T cell responses, such as lymphokine production and cellularproliferation. Protein tyrosine phosphorylation has also been shown tooccur in T cells upon CD28 ligation and it has been demonstrated that aprotein tyrosine kinase inhibitor, herbimycin A, can inhibitCD28-induced IL-2 production (Vandenberghe, P. et al. (1992) J. Exp.Med. 175:951-960; Lu, Y. et al. (1992) J. Immunol. 149:24-29). Thus, toselectively expand a population of CD4⁺ T cells, the CD28 receptormediated pathway can be stimulated by contacting T cells with anactivator of P13K or an agent which stimulates protein tyrosinephosphorylation in the T cell, or both. An activator of PI3K can beidentified based upon its ability to stimulate production of at leastone D-3 phosphoinositide in a T cell. The term “D-3 phosphoinositide” isintended to include derivatives of phosphatidylinositol that arephosphorylated at the D-3 position of the inositol ring and encompassesthe compounds phosphatidylinositol(3)-monophosphate (Ptdlns(3)P),phosphatidylinositol(3,4)-bisphosphate (PtdIns(3,4)P₂), andphosphatidylinositol(3,4,5)-trisphosphate (PtdIns(3,4,5)P₃). Thus, inthe presence of a P13K activator, the amount of a D-3 phosphoinositidein the T cell is increased relative to the amount of the D-3phosphoinositide in the T cell in the absence of the substance.Production of D-3 phosphoinositides (e.g., PtdIns(3)P, PtdIns(3,4)P₂and/or PtdIns(3,4,5)P₃) in a T cell can be assessed by standard methods,such as high pressure liquid chromatography or thin layerchromatography, as discussed above. Similarly, protein tyrosinephosphorylation can be stimulated in a T cell, for example, bycontacting the T cell with an activator of protein tyrosine kinases,such as pervanadate (see O'Shea, J. J. et al. (1992) Proc. Natl. Acad.Sci. USA 89:10306-103101; and Secrist, J. P. (1993) J. Biol. Chem.268:5886-5893). Alternatively, the T cell can be contacted with an agentwhich inhibits the activity of a cellular protein tyrosine phosphatase,such as CD45, to increase the net amount of protein tyrosinephosphorylation in the T cell. Any of these agents can be used to expandan activated population of CD4⁺ T cells in accordance with the methodsdescribed herein.

[0091] Techniques for Expansion of CD8-T Cells

[0092] In order to induce proliferation and expand a population of CD8⁺T cells, an activated population of T cells is stimulated through a 27kD accessory molecule found on activated T cells and recognized by themonoclonal antibody ES5.2D8. As described in Example 9, a population ofCD8⁺ T cells was preferentially expanded by stimulation with an anti-CD3monoclonal antibody and the ES5.2D8 monoclonal antibody. The monoclonalantibody ES5.2D8 was produced by immunization of mice with activatedhuman blood lymphocytes and boosted with recombinant human CTLA4 proteinproduced in E. coli. The ES5.2D8 monoclonal antibody is of the IgG2bisotype and specifically binds to cells transfected with human CTLA4.Hybridomas producing CTLA4-specific antibody were identified byscreening by ELISA against human CTLA4 protein as well as bydifferential FACS against wild type CHO-DG44 cells vs. CHO-105A cells,which are transfected with the human CTLA4 gene. As shown in FIG. 7, theES5.2D8 clone reacts strongly with both activated human T cells andCHO-105A cells but not with CHO-DCA4 cells, indicating that it doesindeed bind to CTLA4. Immunoprecipitation of detergent lysates ofsurface labeled activated human T cells revealed that ES5.2D8 alsoreacts with a 27 kD cell surface protein (FIG. 8). A hybridoma whichproduces the monoclonal antibody ES5.2D8 was deposited on Jun. 4, 1993with the American Type Culture Collection at ATCC Deposit No. HB11374.

[0093] Accordingly, to expand a population of CD8⁺ T cells, an antibody,such as monoclonal antibody ES5.2D8, or other antibody which recognizesthe same 27 kD ligand as ES5.2D8 can be used. As described in Example10, the epitope recognized by the monoclonal antibody ES5.2D8 wasidentified by screening a phage display library (PDL). Antibodies whichbind to the same epitope as the monoclonal antibody ES5.2D8 are withinthe scope of the invention. Such antibodies can be produced byimmunization with a peptide fragment including the epitope or with thenative 27 kD antigen. The term “epitope”, as used herein, refers to theactual structural portion of the antigen that is immunologically boundby an antibody combining site. The term is also used interchangeablywith “antigenic determinant”. A preferred epitope which is bound by anantibody or other ligand which is to be used to stimulate a CD8⁺ T cellpopulation includes or encompasses, an amino acid sequence:

[0094] (Xaa₁)_(n)-Gly-Xaa₂-Trp-Leu-Xaa₃-Xaa₄-Asp(Glu)-(Xaa₅)_(n)(SEQ IDNO: 5),

[0095] wherein Xaa₄ may or may not be present, Xaa₁, Xaa₂, Xaa₃, Xaa₄and Xaa₅ are any amino acid residue and n=0-20, more preferably 0-10,even more preferably 0-5, and most preferably 0-3. In a preferredembodiment, Xaa₂ is Cys, Ile or Leu, Xaa₃ is Leu or Arg and Xaa₄, ifpresent, is Arg, Pro or Phe. As described in Example 10, the monoclonalantibody ES5.2D8, which specifically binds a 27 kD antigen on activatedT cells was used to screen a cDNA library from activated T cells toisolate a clone encoding the antigen. Amino acid sequence analysisidentified the antigen as CD9 (SEQ ID NO: 6). In the native human CD9molecule, epitope defined by phage display library screening is locatedat amino acid residues 31-37 (i.e., G L W L R F D (SEQ ID NO: 7)).Accordingly, Xaa₁ and Xaa₄ are typically additional amino acid residuesfound at either the amino or carboxy side, or both the amino and carboxysides, of the core epitope in the human CD9 (the full-length amino acidsequence of which is shown in SEQ ID NO: 6). It will be appreciated bythose skilled in the art that in the native protein, additionalnon-contiguous amino acid residues may also contribute to theconformational epitope recognized by the antibody. Synthetic peptidesencompassing the epitope can be created which includes other amino acidresidues flanking the core six amino acid residues (i.e., Xaa canalternatively be other amino acid residues than those found in thenative CD9 protein). These flanking amino acid residues can function toalter the properties of the resulting peptide, for example to increasethe solubility, enhance the immunogenicity or promote dimerization ofthe resultant peptide. When the peptide is to be used as an immunogen,one or more charged amino acids (e.g., lysine, arginine) can be includedto increase the solubility of the peptide and/or enhance theimmunogenicity of the peptide. Alternatively, cysteine residues can beincluded to increase the dimerization of the resulting peptide.

[0096] Other embodiments of the invention pertain to expansion of apopulation of CD8⁺ T cells by use of an agent which acts intracellularlyto stimulate a signal in the T cell mediated by ligation of CD9 or otherCD9-associated molecule. It is known that CD9 belongs to the TM4superfamily of cell surface proteins which span the membrane four times(Boucheix, C. et al. (1990) J Biol. Chem. 266, 117-122 and Lanza, F. etal. (1990) J. Biol. Chem. 266, 10638-10645). Other members of the TM4superfamily include CD37, CD53, CD63 and TAPA-1. A role for CD9 ininteracting with GTP binding proteins has been suggested (Sechafer, J.G. and Shaw, A. R. E. (1991) Biochem. Biophys. Res. Commun. 179,401-406). As used herein the term “agent” encompasses chemicals andother pharmaceutical compounds which stimulate a signal in a T cellwithout the requirement for an interaction between a T cell surfacereceptor and a ligand. Thus, this agent does not bind to theextracellular portion of CD9, but rather mimics or induces anintracellular signal (e.g., second messenger) associated with ligationof CD9 or a CD9-associated molecule by an appropriate ligand. Theligands described herein (e.g., monoclonal antibody ES5.2D8) can be usedto identify an intracellular signal(s) associated with T cell expansionmediated by contact of the CD9 antigen or CD9-associated molecule withan appropriate ligand (as described in the Examples) and examining theresultant intracellular signalling that occurs (e.g., protein tyrosinephosphorylation, calcium influx, activation of serine/threonine and/ortyrosine kinases, phosphatidyl inositol metabolism, etc.). An agentwhich enhances an intracellular signal associated with CD9 or aCD9-associated molecule can then be used to expand CD8⁺ T cells.Alternatively, agents (e.g., small molecules, drugs, etc.) can bescreened for their ability to inhibit or enhance T cell expansion usinga system such as that described in the Examples.

[0097] Techniques for Expansion of Antigen Specific T Cells

[0098] In yet another aspect of the invention, methods for expanding apopulation of antigen specific T cells are provided. To produce apopulation of antigen specific T cells, T cells are contacted with anantigen in a form suitable to trigger a primary activation signal in theT cell, i.e., the antigen is presented to the T cell such that a signalis triggered in the T cell through the TCR/CD3 complex. For example, theantigen can be presented to the T cell by an antigen presenting cell inconduction with an MHC molecule. An antigen presenting cell, such as a Bcell, macrophage, monocyte, dendritic cell, Langerhans cell, or othercell which can present antigen to a T cell, can be incubated with the Tcell in the presence of the antigen (e.g., a soluble antigen) such thatthe antigen presenting cell presents the antigen to the T cell.Alternatively, a cell expressing an antigen of interest can be incubatedwith the T cell. For example, a tumor cell expressing tumor-associatedantigens can be incubated with a T cell together to induce atumor-specific response. Similarly, a cell infected with a pathogen,e.g., a virus, which presents antigens of the pathogen can be incubatedwith a T cell. Following antigen specific activation of a population ofT cells, the cells can be expanded in accordance with the methods of theinvention. For example, after antigen specificity has been established,T cells can be expanded by culture with an anti-CD3 antibody and ananti-CD28 antibody according to the methods described herein.

[0099] Production of Antibodies and Coupling of Antibodies to SolidPhase Surfaces

[0100] The term “antibody” as used herein refers to immunoglobulinmolecules and immunologically active portions of immunoglobulinmolecules, i.e., molecules that contain an antigen binding site whichspecifically binds (immunoreacts with) an antigen, such as CD3, CD28.Structurally, the simplest naturally occurring antibody (e.g., IgG)comprises four polypeptide chains, two heavy (H) chains and two light(L) chains inter-connected by disulfide bonds. It has been shown thatthe antigen-binding function of an antibody can be performed byfragments of a naturally-occurring antibody. Thus, these antigen-bindingfragments are also intended to be designated by the term “antibody”.Examples of binding fragments encompassed within the term antibodyinclude (i) an Fab fragment consisting of the VL, VH, CL and CHidomains; (ii) an Fd fragment consisting of the VH and CH1 domains; (iii)an Fv fragment consisting of the VL and VH domains of a single arm of anantibody, (iv) a dAb fragment (Ward et al., (1989) Nature 341:544-546)which consists of a VH domain; (v) an isolated complimentarilydetermining region (CDR); and (vi) an F(ab′)₂ fragment, a bivalentfragment comprising two Fab fragments linked by a disulfide bridge atthe hinge region. Furthermore, although the two domains of the Fvfragment are coded for by separate genes, a synthetic linker can be madethat enables them to be made as a single protein chain (known as singlechain Fv (scFv); Bird et al. (1988) Science 242:423-426; and Huston etal. (1988) PNAS 85:5879-5883) by recombinant methods. Such single chainantibodies are also encompassed within the term “antibody”. Preferredantibody fragments for use in T cell expansion are those which arecapable of crosslinking their target antigen, e.g., bivalent fragmentssuch as F(ab′)₂ fragments. Alternatively, an antibody fragment whichdoes not itself crosslink its target antigen (e.g., a Fab fragment) canbe used in conjunction with a secondary antibody which serves tocrosslink the antibody fragment, thereby crosslinking the targetantigen. Antibodies can be fragmented using conventional techniques asdescribed herein and the fragments screened for utility in the samemanner as described for whole antibodies. An antibody of the inventionis further intended to include bispecific and chimeric molecules havinga desired binding portion (e.g., CD28).

[0101] The language “a desired binding specificity for an epitope”, aswell as the more general language “an antigen binding site whichspecifically binds (immunoreacts with)”, refers to the ability ofindividual antibodies to specifically immunoreact with a T cell surfacemolecule, e.g., CD28. That is, it refers to a non-random bindingreaction between an antibody molecule and an antigenic determinant ofthe T cell surface molecule. The desired binding specificity istypically determined from the reference point of the ability of theantibody to differentially bind the T cell surface molecule and anunrelated antigen, and therefore distinguish between two differentantigens, particularly where the two antigens have unique epitopes. Anantibody which binds specifically to a particular epitope is referred toas a “specific antibody”.

[0102] “Antibody combining site”, as used herein, refers to thatstructural portion of an antibody molecule comprised of a heavy andlight chain variable and hypervariable regions that specifically binds(immunoreacts with) antigen. The term “immunoreact” or “reactive with”in its various forms is used herein to refer to binding between anantigenic determinant-containing molecule and a molecule containing anantibody combining site such as a whole antibody molecule or a portionthereof.

[0103] Although soluble forms of antibodies may be used to activate Tcells, it is preferred that the anti-CD3 antibody be immobilized on asolid phase surface (e.g., beads). An antibody can be immobilizeddirectly or indirectly by, for example, by a secondary antibody, to asolid surface, such as a tissue culture flask or bead. As anillustrative embodiment, the following is a protocol for immobilizing ananti-CD3 antibody on beads. It should be appreciated that the sameprotocol can be used to immobilize other antibodies or fragments thereof(e.g., an anti-CD28 antibody), and Ig fusion proteins, such as B7Igfusion proteins, to beads. The same protocol can also be used toimmobilize more than one antibody, or an antibody and another molecule,such as a fusion protein, to the the solid phase surface.

Protocols

[0104] I. Pre-absorbing Goat anti-mouse IgG with OKT-3

[0105] A) BioMag Goat anti-Mouse IgG (Advanced Magnetics, Inc., catalognumber 8-4340D) is incubated with at least 200 μg of OKT-3 per 5×10⁸magnetic particles in PBS for 1 hour at 5° C.

[0106] B) Particles are washed three time in PBS with the aid of amagnetic separation unit.

[0107] Note: Advanced Magnetics also has an anti-Human CD3 directlyconjugated (Catalog number 8-4703N) which will induce T-cellstimulation.

[0108] II. Pre-labeling Lymphocytes with OKT-3

[0109] A) 1×10⁶ cells (PBMC) are incubated in PBS with 10 μg/ml of OKT-3for 15 minutes at room temperature.

[0110] B) Cells are washed twice with PBS.

[0111] III. Binding Magnetic Particles to PBMC for Stimulation

[0112] A) PBMC surface labeled with OKT-3 are cultured with Goatanti-Mouse IgG (see above) at one bead per cell following a 30 minuteincubation at 20° C. with gentle agitation.

[0113] B) Goat anti-Mouse IgG beads which were previously absorbed toOKT-3 are incubated with PBMC (1:1) for 30 minutes at 20° C. with gentleagitation and cultured.

[0114] IV. Binding Magnetic Particles to PBMC for Separation Same asabove (Part III) except the bead to cell ratio is increased to 20:1rather than 1:1.

[0115] Alternatively, antibodies can be coupled to a solid phasesurface, e.g., beads by crosslinking via covalent modification usingtosyl linkage. In one method, an antibody such as OKT3 is in 0.05Mborate buffer, pH 9.5 and added to tosyl activated magnetic immunobeads(Dynal Inc., Great Neck, N.Y.) according to the manufacturer'sinstructions. After a 24 hr incubation at 22° C., the beads arecollected and washed extensively. It is not mandatory thatimmunomagnetic beads be used, as other methods are also satisfactory.

[0116] To practice the method of the invention, a source of T cells isobtained from a subject. The term subject is intended to include livingorganisms in which an immune response can be elicited, e.g., mammals.Examples of subjects include humans, dogs, cats, mice, rats, andtransgenic species thereof. T cells can be obtained from a number ofsources, including peripheral blood leukocytes, bone marrow, lymph nodetissue, spleen tissue, and tumors. Preferably, peripheral bloodleukocytes are obtained from an individual by leukopheresis. To isolateT cells from peripheral blood leukocytes, it may be necessary to lysethe red blood cells and separate peripheral blood leukocytes frommonocytes by, for example, centrifugation through a PERCOLL™ gradient. Aspecific subpopulation of T cells, such as CD28⁺, CD4⁺, CD8⁺, CD28RA⁺,and CD28RO⁺ T cells, can be further isolated by positive or negativeselection techniques. For example, negative selection of a T cellpopulation can be accomplished with a combination of antibodies directedto surface markers unique to the cells negatively selected. A preferredmethod is cell sorting via negative magnetic immunoadherence whichutilizes a cocktail of monoclonal antibodies directed to cell surfacemarkers present on the cells negatively selected. For example, toisolate CD4⁺ cells, a monoclonal antibody cocktail typically includesantibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8. Additionalmonoclonal antibody cocktails are provided in Table 1.

[0117] The process of negative selection results in an essentiallyhomogenous population of CD28⁺, CD4⁺ or CD8⁺ T cells. The T cells can beactivated as described herein, such as by contact with a anti-CD3antibody immobilized on a solid phase surface or an anti-CD2 antibody,or by contact with a protein kinase C activator (e.g., bryostatin) inconjunction with a calcium ionophore. To stimulate an accessory moleculeon the surface of the T cells, a ligand which binds the accessorymolecule is employed. For example, a population of CD4⁺ cells can becontacted with an anti-CD3 antibody and an anti-CD28 antibody, underconditions appropriate for stimulating proliferation of the T cells.Similarly, to stimulate proliferation of CD8⁺ T cells, an anti-CD3antibody and the monoclonal antibody ES5.2D8 can be used. Conditionsappropriate for T cell culture include an appropriate media (e.g.,Minimal Essential Media or RPMI Media 1640) which may contain factorsnecessary for proliferation and viability, including animal serum (e.g.,fetal bovine serum) and antibiotics (e.g., penicillin streptomycin). TheT cells are maintained under conditions necessary to support growth, forexample an appropriate temperature (e.g., 37° C.) and atmosphere (e.g.,air plus 5% CO₂).

[0118] The primary activation signal and the costimulatory signal forthe T cell can be provided by different protocols. For example, theagents providing each signal can be in solution or coupled to a solidphase surface. When coupled to a solid phase surface, the agents can becoupled to the same solid phase surface (i.e., in “cis” formation) or toseparate surfaces (i.e., in “trans” formation). Alternatively, one agentcan be coupled to a solid phase surface and the other agent in solution.In one embodiment, the agent providing the costimulatory signal is boundto a cell surface and the agent providing the primary activation signalis in solution or coupled to a solid phase surface. In a preferredembodiment, the two agents are coupled to beads, either to the samebead, i.e., in “cis”, or to separate beads, i.e., in “trans”.Alternatively, the agent providing the primary activation signal is ananti-CD3 antibody and the agent providing the costimulatory signal is ananti-CD28 antibody; both agents are coupled to the same beads. In thisembodiment, it has been determined that the optimal ratio of eachantibody bound to the beads for CD4⁺ T cell expansion and T cell growthfor up to at least 50 days is a 1:1 ratio. However, ratios from 1:9 to9:1 can also be used to stimulate CD2⁺ T cell expansion. The ratio ofanti-CD3 and anti-CD28 coated (with a ratio of 1:1 of each antibody)beads to T cells that yield T cell expansion can vary from 1:3 to 3: 1,with the optimal ratio being 3:1 beads per T cell. Moreover, it has beendetermined that when T cells are expanded under these conditions, theyremain polyclonal.

[0119] To maintain long term stimulation of a population of T cellsfollowing the initial activation and stimulation, it is necessary toseparate the T cells from the activating stimulus (e.g., the anti-CD3antibody) after a period of exposure. The T cells are maintained incontact with the co-stimulatory ligand throughout the culture term. Therate of T cell proliferation is monitored periodically (e.g., daily) by,for example, examining the size or measuring the volume of the T cells,such as with a Coulter Counter. A resting T cell has a mean diameter ofabout 6.8 microns. Following the initial activation and stimulation andin the presence of the stimulating ligand, the T cell mean diameter willincrease to over 12 microns by day 4 and begin to decrease by about day6. When the mean T cell diameter decreases to approximately 8 microns,the T cells are reactivated and restimulated to induce furtherproliferation of the T cells. Alternatively, the rate of T cellproliferation and time for T cell restimulation can be monitored byassaying for the presence of cell surface molecules, such as B7-1, B7-2,which are induced on activated T cells. As described in Example 5, itwas determined that CD4⁺ T cells do not initially express the B7-1receptor, and that with culture, expression is induced. Further, theB7-1 expression was found to be transient, and could be re-induced withrepeated anti-CD3 restimulation. Accordingly, cyclic changes in B7-1expression can be used as a means of monitoring T cell proliferation;where decreases in the level of B7-1 expression, relative to the levelof expression following an initial or previous stimulation or the levelof expression in an unstimulated cell, indicates the time forrestimulation.

[0120] For inducing long term stimulation of a population of CD4⁺ orCD8⁺ T cells, it may be necessary to reactivate and restimulate the Tcells with a anti-CD3 antibody and an anti-CD28 antibody or monoclonalantibody ES5.2D8 several times to produce a population of CD4⁺ or CD8⁺cells increased in number from about 10- to about 1,000-fold theoriginal T cell population. Using this methodology, it is possible toget increases in a T cell population of from about 100- to about100,000-fold an original resting T cell population. Moreover, asdescribed in Example 6, T cells expanded by the method of the inventionsecrete high levels of cytokines (e.g., IL-2, IFNγ, IL-4, GM-CSF andTNFα) into the culture supernatants. For example, as compared tostimulation with IL-2, CD4⁺ T cells expanded by use of anti-CD3 andanti-CD28 costimulation secrete high levels of GM-CSF and TNFA into theculture medium. These cytokines can be purified from the culturesupernatants or the supernatants can be used directly for maintainingcells in culture. Similarly, the T cells expanded by the method of theinvention together with the culture supernatant and cytokines can beadministered to support the growth of cells in vivo. For example, inpatients with tumors, T cells can be obtained from the individual,expanded in vitro and the resulting T cell population and supernatant,including cytokines such as TNFα, can be readministered to the patientto augment T cell growth in vivo.

[0121] The invention also provides methods for expanding a population ofT cells by a factor of about 10log₁₀ to about 12log₁₀, while maintainingthe polyclonality of the population of T cells, as described in Example18. In this embodiment, the T cells are stimulated with anti-CD3 andanti-CD28 coated beads and IL-2 is added to the culture at about day 49of the culture. It is important to replenish the culture medium withIL-2, since it has been shown that the amount of IL-2 produced by Tcells in long term culture decreases with time. This can for example beseen in FIG. 28, which shows that T cells stimulated with anti-CD3 andanti-CD28 in trans secrete progressively less IL-2 with time in cultureas can be seen by comparing the amount of IL-2 secreted on day 1, day12, and on day 21 of the culture.

[0122] The amount of IL-2 that should be added to the T cell culture toobtain expansion of the order of about 10log₁₀ to about 12log₁₀ can bedetermined without undue experimentation. In preliminary experiments,the amount of IL-2 secreted and the proliferation of the T cells aremeasured during long term proliferation assays (see Example 20). Thus,it is possible to determine the concentrations of IL-2 required foroptimal T cell proliferation and the amount of IL-2 that should be addedto the culture once proliferation of the T cells has slowed down.Moreover, amounts of IL-2 required for T cell growth are well known inthe art and can thus easily be determined.

[0123] In one embodiment of the invention pertaining to polyclonalexpansion of T cells to about 10log₁₀ to about 12log₁₀, the amount ofIL-2 in the culture medium is monitored and IL-2 is added to the culturewhen the level of IL-2 in the supernatant is lower than the amount ofIL-2 sufficient to maintain proliferation, preferably optimalproliferation, of the T cells. The phrase “IL-2 is added in amountssufficient to maintain proliferation” of the T cells, e.g., CD4⁺ Tcells, refers to the amount of IL-2 that is added to obtain a finalconcentration of IL-2 in the supernatant that corresponds to the amountdetermined to allow for the proliferation of the T cells. The optimalamount of IL-2 that is required can be determined as described in theprevious paragraph and in Example 20. In another embodiment, IL-2 isadded from the first day of the culture, and added every other day ofthe culture in amounts sufficient to maintain proliferation of the Tcells. Alternatively, IL-2 can be added to the cultures to obtain afinal concentration of about 100 U/ml and added every other day to theculture, such as every second or third day, when new medium is added tothe cell culture.

[0124] It is also possible to obtain expansion of T lymphocytes by afactor from about 10log₁₀ to about 12log₁₀ by incubating the T cellswith beads coated with anti-CD3 antibody, such as OKT3 and a stimulatoryform of B7-2, such as B7-2Ig and the addition of IL-2 in amountssufficient to maintain proliferation of the T cells.

[0125] Although the antibodies used in the methods described herein canbe readily obtained from public sources, such as the ATCC, antibodies toT cell surface accessory molecules, the CD3 complex, or CD2 can beproduced by standard techniques. Methodologies for generating antibodiesfor use in the methods of the invention are described in further detailbelow.

[0126] Specific Methodology for Antibody Production

[0127] A. The Immunogen.

[0128] The term “immunogen” is used herein to describe a compositioncontaining a peptide or protein as an active ingredient used for thepreparation of antibodies against an antigen (e.g., CD3, CD28). When apeptide or protein is used to induce antibodies it is to be understoodthat the peptide can be used alone, or linked to a carrier as aconjugate, or as a peptide polymer.

[0129] To generate suitable antibodies, the immunogen should contain aneffective, immunogenic amount of a peptide or protein, optionally as aconjugate linked to a carrier. The effective amount of peptide per unitdose depends, among other things, on the species of animal inoculated,the body weight of the animal and the chosen immunization regimen as iswell known in the art. The immunogen preparation will typically containpeptide concentrations of about 10 micrograms to about 500 milligramsper immunization dose, preferably about 50 micrograms to about 50milligrams per dose. An immunization preparation can also include anadjuvant as part of the diluent. Adjuvants such as complete Freund'sadjuvant (CFA), incomplete Freund's adjuvant (IFA) and alum arematerials well known in the art, and are available commercially fromseveral sources.

[0130] Those skilled in the art will appreciate that, instead of usingnatural occurring forms of the antigen (e.g., CD3, CD28) forimmunization, synthetic peptides can alternatively be employed towardswhich antibodies can be raised for use in this invention. Both solubleand membrane bound forms of the protein or peptide fragments aresuitable for use as an immunogen and can also be isolated byimmunoaffinity purification as well. A purified form of protein, such asmay be isolated as described above or as known in the art, can itself bedirectly used as an immunogen, or alternatively, can be linked to asuitable carrier protein by conventional techniques, including bychemical coupling means as well as by genetic engineering using a clonedgene of the protein. The purified protein can also be covalently ornoncovalently modified with non-proteinaceous materials such as lipidsor carbohydrates to enhance immunogenecity or solubility. Alternatively,a purified protein can be coupled with or incorporated into a viralparticle, a replicating virus, or other microorganism in order toenhance immunogenicity. The protein may be, for example, chemicallyattached to the viral particle or microorganism or an immunogenicportion thereof.

[0131] In an illustrative embodiment, a purified CD28 protein, or apeptide fragment thereof (e.g., produced by limited proteolysis orrecombinant DNA techniques) is conjugated to a carrier which isimmunogenic in animals. Preferred carriers include proteins such asalbumins, serum proteins (e.g., globulins and lipoproteins), andpolyamino acids. Examples of useful proteins include bovine serumalbumin, rabbit serum albumin, thyroglobulin, keyhole limpet hemocyanin,egg ovalbumin and bovine gamma-globulins. Synthetic polyamino acids suchas polylysine or polyarginine are also useful carriers. With respect tothe covalent attachment of CD28 protein or peptide fragments to asuitable immunogenic carrier, there are a number of chemicalcross-linking agents that are known to those skilled in the art.Preferred cross-linking agents are heterobifunctional cross-linkers,which can be used to link proteins in a stepwise manner. A wide varietyof heterobifunctional cross-linkers are known in the art, includingsuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC),m-Maleimidobenzoyl-N-hydroxysuccinimide ester (MBS); N-succinimidyl(4-iodoacetyl) aminobenzoate (SIAB), succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimidehydrochloride (EDC);4-succinimidyl-oxycarbonyl-a-methyl-a-(2-pyridyldithio)-tolune (SMPT),N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP), succinimidyl6-[3-(2-pyridyldithio) propionate] hexanoate (LC-SPDP).

[0132] It may also be desirable to simply immunize an animal with wholecells which express a protein of interest (e.g., CD28) on their surface.Various cell lines can be used as immunogens to generate monoclonalantibodies to an antigen, including, but not limited to T cells. Forexample, peripheral blood T cells can be obtained from a subject whichconstituitively express CD28, but can be activated in vitro withanti-CD3 antibodies, PHA or PMA. Alternatively, an antigen specific(e.g., alloreactive) T cell clone can be activated to express CD28 bypresentation of antigen, together with a costimulatory signal, to the Tcell. Whole cells that can be used as immunogens to produce CD28specific antibodies also include recombinant transfectants. For example,COS and CHO cells can be reconstituted by transfection with a CD28 cDNAto produce cells expressing CD28 on their surface. These transfectantcells can then be used as immunogens to produce anti-CD28 antibodies.Other examples of transfectant cells are known, particularly eukaryoticcells able to glycosylate the CD28 protein, but any procedure that worksto express transfected CD28 genes on the cell surface could be used toproduce the whole cell immunogen.

[0133] Alternative to a CD28-expressing cell or an isolated CD28protein, peptide fragments of CD28 or other surface antigen such as CD9can be used as immunogens to generate antibodies. For example, the CD9epitope bound by the ES5.2D8 monoclonal antibody comprises an amino acidsequence: (Xaa₁)_(n)-Gly-Xaa₂-Trp-Leu-Xaa₃-Xaa₄-Asp(Glu)-(Xaa₅)_(n) (SEQID NO: 5), wherein Xaa₄ may or may not be present, Xaa₁, Xaa₂, Xaa₃,Xaa₄ and Xaa₅ are any amino acid residue and n=0-20, more preferably0-10, even more preferably 0-5, and most preferably 0-3. In a preferredembodiment, Xaa2 is Cys, Ile or Leu, Xaa₃ is Leu or Arg and Xaa₄, ifpresent, is Arg, Pro or Phe. Thus, a peptide having the amino acidsequence of SEQ ID NO: 5 can be used as an immunogen. Accordingly, theinvention further encompasses an isolated CD9 peptide comprising anamino acid sequence:(Xaa₁)_(n)-Gly-Xaa₂-Trp-Leu-Xaa₃-Xaa₄-Asp(Glu)-(Xaa5)n (SEQ ID NO: 5),wherein Xaa₄ may or may not be present, Xaa₁, Xaa₂, Xaa₃, Xaa₄ and Xaa₅are any amino acid residue and n=0-20, more preferably 0-10, even morepreferably 0-5, and most preferably 0-3. In a preferred embodiment, Xaa₂is Cys, Ile or Leu, Xaa₃ is Leu or Arg and Xaa₄, if present, is Arg, Proor Phe. Alternatively, it has been found that the ES5.2D8 monoclonalantibody cross-reacts with a number of other peptide sequences(determined by phage display technology as described in Example 3).Examples of these other peptide sequences are shown below:

[0134] 2D8#2(SEQ ID NO: 8) H Q F C D H W G C W L L R E T H I F T P 2D8#4

[0135] (SEQ ID NO: 8) H Q F C D H W G C W L L R E T H I F T P

[0136] 2D8#10(SEQ ID NO: 8) H Q F C D H W G C W L L R E T H I F T P

[0137] 2D8#6(SEQ ID NO: 9) L R L V L E D P G I W L R P D Y F F P A

[0138] G C W L L R E (phage 2D8#2,4, 10; SEQ ID NO: 10)

[0139] G I W L R P D(phage 2D8#6;SEQ ID NO: 11)

[0140] G L W L R F D (CD9 sequence; SEQ ID NO: 7)

[0141] Any of these peptides, or other peptides containing a stretch ofseven amino acids bracketed in bold type (representing the epitope boundby the antibody) possibly flanked by alternative amino acid residues,can also be used as immunogens to produce an antibody for use in themethods of the invention and are encompassed by the invention. For useas immunogens, peptides can be modified to increase solubility and/orenhance immunogenicity as described above.

[0142] B. Polyclonal Antibodies.

[0143] Polycolonal antibodies to a purified protein or peptide fragmentthereof can generally be raised in animals by multiple subcutaneous (sc)or intraperitoneal (ip) injections of an appropriate immunogen, such asthe extracellular domain of the protein, and an adjuvant. A polyclonalantisera can be produced, for example, as described in Lindsten, T. etal. (1993) J. Immunol. 151:3489-3499. In an illustrative embodiment,animals are typically immunized against the immunogenic protein, peptideor derivative by combining about 1 μg to 1 mg of protein with Freund'scomplete adjuvant and injecting the solution intradermally at multiplesites. One month later the animals are boosted with ⅕ to {fraction(1/10)} the original amount of immunogen in Freund's complete adjuvant(or other suitable adjuvant) by subcutaneous injection at multiplesites. Seven to 14 days later, the animals are bled and the serum isassayed for anti-protein or peptide titer (e.g., by ELISA). Animals areboosted until the titer plateaus. Also, aggregating agents such as alumcan be used to enhance the immune response.

[0144] Such mammalian-produced populations of antibody molecules arereferred to as “polyclonal” because the population comprises antibodieswith differing immunospecificities and affinities for the antigen. Theantibody molecules are then collected from the mammal (e.g., from theblood) and isolated by well known techniques, such as protein Achromatography, to obtain the IgG fraction. To enhance the specificityof the antibody, the antibodies may be purified by immunoaffinitychromatography using solid phase-affixed immunogen. The antibody iscontacted with the solid phase-affixed immunogen for a period of timesufficient for the immunogen to immunoreact with the antibody moleculesto form a solid phase-affixed immunocomplex. The bound antibodies areseparated from the complex by standard techniques.

[0145] C. Monoclonal Antibodies.

[0146] The term “monoclonal antibody” or “monoclonal antibodycomposition”, as used herein, refers to a population of antibodymolecules that contain only one species of an antigen binding sitecapable of immunoreacting with a particular epitope of an antigen. Amonoclonal antibody composition thus typically displays a single bindingaffinity for a particular protein with which it immunoreacts.Preferably, the monoclonal antibody used in the subject method isfurther characterized as immunoreacting with a protein derived fromhumans.

[0147] Monoclonal antibodies useful in the methods of the invention aredirected to an epitope of an antigen(s) on T cells, such that complexformation between the antibody and the antigen (also referred to hereinas ligation) induces stimulation and T cell expansion. A monoclonalantibody to an epitope of an antigen (e.g., CD3, CD28) can be preparedby using a technique which provides for the production of antibodymolecules by continuous cell lines in culture. These include but are notlimited to the hybridoma technique originally described by Kohler andMilstein (1975, Nature 256:495-497), and the more recent human B cellhybridoma technique (Kozbor et al. (1983) Immunol Today 4:72),EBV-hybridoma technique (Cole et al. (1985), Monoclonal Antibodies andCancer Therapy, Alan R. Liss, Inc., pp. 77-96), and trioma techniques.Other methods which can effectively yield monoclonal antibodies usefulin the present invention include phage display techniques (Marks et al.(1992) J Biol Chem 16007-16010).

[0148] In one embodiment, the antibody preparation applied in thesubject method is a monoclonal antibody produced by a hybridoma cellline. Hybridoma fusion techniques were first introduced by Kohler andMilstein (Kohler et al. Nature (1975) 256:495-97; Brown et al. (1981) J.Immunol 127:539-46; Brown et al. (1980) J Biol Chem 255:4980-83; Yeh etal. (1976) PNAS 76:2927-31; and Yeh et al. (1982) Int. J. Cancer29:269-75). Thus, the monoclonal antibody compositions of the presentinvention can be produced by the following method, which comprises thesteps of:

[0149] (a) Immunizing an animal with a protein (e.g., CD28) or peptidethereof. The immunization is typically accomplished by administering theimmunogen to an immunologically competent mammal in an immunologicallyeffective amount, i.e., an amount sufficient to produce an immuneresponse. Preferably, the mammal is a rodent such as a rabbit, rat ormouse. The mammal is then maintained for a time period sufficient forthe mammal to produce cells secreting antibody molecules thatimmunoreact with the immunogen. Such immunoreaction is detected byscreening the antibody molecules so produced for immunoreactivity with apreparation of the immunogen protein. Optionally, it may be desired toscreen the antibody molecules with a preparation of the protein in theform in which it is to be detected by the antibody molecules in anassay, e.g., a membrane-associated form of the antigen (e.g., CD28).These screening methods are well known to those of skill in the art,e.g., enzyme-linked immunosorbent assay (ELISA) and/or flow cytometry.

[0150] (b) A suspension of antibody-producing cells removed from eachimmunized mammal secreting the desired antibody is then prepared. Aftera sufficient time, the mouse is sacrificed and somaticantibody-producing lymphocytes are obtained. Antibody-producing cellsmay be derived from the lymph nodes, spleens and peripheral blood ofprimed animals. Spleen cells are preferred, and can be mechanicallyseparated into individual cells in a physiologically tolerable mediumusing methods well known in the art. Mouse lymphocytes give a higherpercentage of stable fusions with the mouse myelomas described below.Rat, rabbit and frog somatic cells can also be used. The spleen cellchromosomes encoding desired immunoglobulins are immortalized by fusingthe spleen cells with myeloma cells, generally in the presence of afusing agent such as polyethylene glycol (PEG). Any of a number ofmyeloma cell lines may be used as a fusion partner according to standardtechniques; for example, the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 orSp2/O-Ag14 myeloma lines. These myeloma lines are available from theAmerican Type Culture Collection (ATCC), Rockville, Md.

[0151] The resulting cells, which include the desired hybridomas, arethen grown in a selective medium, such as HAT medium, in which unfusedparental myeloma or lymphocyte cells eventually die. Only the hybridomacells survive and can be grown under limiting dilution conditions toobtain isolated clones. The supernatants of the hybridomas are screenedfor the presence of antibody of the desired specificity, e.g., byimmunoassay techniques using the antigen that has been used forimmunization. Positive clones can then be subcloned under limitingdilution conditions and the monoclonal antibody produced can beisolated. Various conventional methods exist for isolation andpurification of the monoclonal antibodies so as to free them from otherproteins and other contaminants. Commonly used methods for purifyingmonoclonal antibodies include ammonium sulfate precipitation, ionexchange chromatography, and affinity chromatography (see, e.g., Zola etal. in Monoclonal Hybridoma Antibodies: Techniques And Applications,Hurell (ed.) pp. 51-52 (CRC Press 1982)). Hybridomas produced accordingto these methods can be propagated in vitro or in vivo (in ascitesfluid) using techniques known in the art.

[0152] Generally, the individual cell line may be propagated in vitro,for example in laboratory culture vessels, and the culture mediumcontaining high concentrations of a single specific monoclonal antibodycan be harvested by decantation, filtration or centrifugation.Alternatively, the yield of monoclonal antibody can be enhanced byinjecting a sample of the hybridoma into a histocompatible animal of thetype used to provide the somatic and myeloma cells for the originalfusion. Tumors secreting the specific monoclonal antibody produced bythe fused cell hybrid develop in the injected animal. The body fluids ofthe animal, such as ascites fluid or serum, provide monoclonalantibodies in high concentrations. When human hybridomas orEBV-hybridomas are used, it is necessary to avoid rejection of thexenograft injected into animals such as mice. Immunodeficient or nudemice may be used or the hybridoma may be passaged first into irradiatednude mice as a solid subcutaneous tumor, cultured in vitro and theninjected intraperitoneally into pristane primed, irradiated nude micewhich develop ascites tumors secreting large amounts of specific humanmonoclonal antibodies.

[0153] Media and animals useful for the preparation of thesecompositions are both well known in the art and commercially availableand include synthetic culture media, inbred mice and the like. Anexemplary synthetic medium is Dulbecco's minimal essential medium (DMEM;Dulbecco et al. (1959) Virol. 8:396) supplemented with 4.5 gm/1 glucose,20 mM glutamine, and 20% fetal caf serum. An exemplary inbred mousestrain is the Balb/c.

[0154] D. Combinatorial Antibodies.

[0155] Monoclonal antibody compositions of the invention can also beproduced by other methods well known to those skilled in the art ofrecombinant DNA technology. An alternative method, referred to as the“combinatorial antibody display” method, has been developed to identifyand isolate antibody fragments having a particular antigen specificity,and can be utilized to produce monoclonal antibodies (for descriptionsof combinatorial antibody display see e.g., Sastry et al. (1989) PNAS86:5728; Huse et al. (1989) Science 246:1275; and Orlandi et al. (1989)PNAS 86:3833). After immunizing an animal with an appropriate immunogen(e.g., CD3, CD28) as described above, the antibody repertoire of theresulting B-cell pool is cloned. Methods are generally known fordirectly obtaining the DNA sequence of the variable regions of a diversepopulation of immunoglobulin molecules by using a mixture of oligomerprimers and PCR. For instance, mixed oligonucleotide primerscorresponding to the 5′ leader (signal peptide) sequences and/orframework 1 (FRI) sequences, as well as primer to a conserved 3′constant region primer can be used for PCR amplification of the heavyand light chain variable regions from a number of murine antibodies(Larrick et al. (1991) Biotechniques 11:152-156). A similar strategy canalso been used to amplify human heavy and light chain variable regionsfrom human antibodies (Larrick et al. (1991) Methods: Companion toMethods in Enzymology 2:106-110).

[0156] In an illustrative embodiment, RNA is isolated from activated Bcells of, for example, peripheral blood cells, bone marrow, or spleenpreparations, using standard protocols (e.g., U.S. Pat. No. 4,683,202;Orlandi, et al. PNAS (1989) 86:3833-3837; Sastry et al., PNAS (1989)86:5728-5732; and Huse et al. (1989) Science 246:1275-1281.)First-strand cDNA is synthesized using primers specific for the constantregion of the heavy chain(s) and each of the K and X light chains, aswell as primers for the signal sequence. Using variable region PCRprimers, the variable regions of both heavy and light chains areamplified, each alone or in combinantion, and ligated into appropriatevectors for further manipulation in generating the display packages.Oligonucleotide primers useful in amplification protocols may be uniqueor degenerate or incorporate inosine at degenerate positions.Restriction endonuclease recognition sequences may also be incorporatedinto the primers to allow for the cloning of the amplified fragment intoa vector in a predetermined reading frame for expression.

[0157] The V-gene library cloned from the immunization-derived antibodyrepertoire can be expressed by a population of display packages,preferably derived from filamentous phage, to form an antibody displaylibrary. Ideally, the display package comprises a system that allows thesampling of very large variegated antibody display libraries, rapidsorting after each affinity separation round, and easy isolation of theantibody gene from purified display packages. In addition tocommercially available kits for generating phage display libraries(e.g., the Pharmacia Recombinant Phage Antibody System, catalog no.27-9400-01; and the Stratagene SurfZAP™ phage display kit, catalog no.240612), examples of methods and reagents particularly amenable for usein generating a variegated antibody display library can be found in, forexample, Ladner et al. U.S. Pat. No. 5,223,409; Kang et al.International Publication No. WO 92/18619; Dower et al. InternationalPublication No. WO 91/17271; Winter et al. International Publication WO92/20791; Markland et al. International Publication No. WO 92/15679;Breitling et al. International Publication WO 93/01288; McCafferty etal. International Publication No. WO 92/01047; Garrard et al.International Publication No. WO 92/09690; Ladner et al. InternationalPublication No. WO 90/02809; Fuchs et al. (1991) Bio/Technology9:1370-1372; Hay et al. (1992) Hum Antibod Hybridomas 3:81-85; Huse etal. (1989) Science 246:1275-1281; Griffths et al. (1993) EMBO J12:725-734; Hawkins et al. (1992) J Mol Biol 226:889-896; Clackson etal. (1991) Nature 352:624-628; Gram et al. (1992) PNAS 89:3576-3580;Garrad et al. (1991) Bio/Technology 9:1373-1377; Hoogenboom et al.(1991) Nuc Acid Res 19:4133-4137; and Barbas et al. (1991) PNAS88:7978-7982.

[0158] In certain embodiments, the V region domains of heavy and lightchains can be expressed on the same polypeptide, joined by a flexiblelinker to form a single-chain Fv fragment, and the scFV genesubsequently cloned into the desired expression vector or phage genome.As generally described in McCafferty et al., Nature (1990) 348:552-554,complete V_(H) and V_(L) domains of an antibody, joined by a flexible(Gly₄-Ser)₃ linker can be used to produce a single chain antibody whichcan render the display package separable based on antigen affinity.Isolated scFV antibodies immunoreactive with the antigen cansubsequently be formulated into a pharmaceutical preparation for use inthe subject method.

[0159] Once displayed on the surface of a display package (e.g.,filamentous phage), the antibody library is screened with the protein,or peptide fragment thereof, to identify and isolate packages thatexpress an antibody having specificity for the protein. Nucleic acidencoding the selected antibody can be recovered from the display package(e.g., from the phage genome) and subcloned into other expressionvectors by standard recombinant DNA techniques.

[0160] E. Hybridomas and Methods of Preparation.

[0161] Hybridomas useful in the present invention are thosecharacterized as having the capacity to produce a monoclonal antibodywhich will specifically immunoreact with an antigen of interest (e.g.,CD3, CD28). Methods for generating hybridomas that produce, e.g.,secrete, antibody molecules having a desired immunospecificity, e.g.,having the ability to immunoreact with the CD28 antigen, and/or anidentifiable epitope of CD28 are well known in the art. Particularlyapplicable is the hybridoma technology described by Niman et al. (1983)PNAS 80:4949-4953; and by Galfre et al. (1981) Meth. Enzymol. 73:3-46.

[0162] Uses of the Methods of the Invention

[0163] The method of this invention can be used to selectively expand apopulation of CD28⁺, CD4⁺, CD8⁺, CD28RA⁺, or CD28RO⁺ T cells for use inthe treatment of infectious disease, cancer and immunotherapy. As aresult of the method described herein, a population of T cells which ispolyclonal with respect to antigen reactivity, but essentiallyhomogeneous with respect to either CD4⁺ or CD8⁺ can be produced. Inaddition, the method allows for the expansion of a population of T cellsin numbers sufficient to reconstitute an individual's total CD4⁺ or CD8⁺T cell population (the population of lymphocytes in an individual isapproximately 10¹¹). The resulting T cell population can be geneticallytransduced and used for immunotherapy or can be used in methods of invitro analyses of infectious agents. For example, a population oftumor-infiltrating lymphocytes can be obtained from an individualafflicted with cancer and the T cells stimulated to proliferate tosufficient numbers. The resulting T cell population can be geneticallytransduced to express tumor necrosis factor (TNF) or other factor andrestored to the individual.

[0164] One particular use for the CD4⁺ T cells expanded by the method ofthe invention is in the treatment of HIV infection in an individual.Prolonged infection with HIV eventually results in a marked decline inthe number of CD4⁺ T lymphocytes. This decline, in turn, causes aprofound state of immunodeficiency, rendering the patient susceptible toan array of life threatening opportunistic infections. Replenishing thenumber of CD4⁺ T cells to normal levels may be expected to restoreimmune function to a significant degree. Thus, the method describedherein provides a means for selectively expanding CD4⁺ T cells tosufficient numbers to reconstitute this population in an HIV infectedpatient. It may also be necessary to avoid infecting the T cells duringlong-term stimulation or it may desirable to render the T cellspermanently resistant to HIV infection. There are a number of techniquesby which T cells may be rendered either resistant to HIV infection orincapable of producing virus prior to restoring the T cells to theinfected individual. For example, one or more anti-retroviral agents canbe cultured with CD4⁺ T cells prior to expansion to inhibit HIVreplication or viral production (e.g., drugs that target reversetranscriptase and/or other components of the viral machinery, see e.g.,Chow et al. (1993) Nature 361, 650-653).

[0165] Several methods can be used to genetically transduce T cells toproduce molecules which inhibit HIV infection or replication. Forexample, in one embodiment, T cells can be genetically transduced toproduce transdominant inhibitors, which are mutated, nonfunctional formsof normal HIV gene products. Transdominant inhibitors function tooligomerize or compete for binding with the wild type HIV proteins.Several transdominant inhibitors have been derived from HIV proteinsincluding tat, rev, and gag. The function of tat is to enhance thetranscription of viral genes by binding to the trans activation responseelement (tar) found in the promoter region of most HIV genes. Rev,through binding to the rev response element (RRE) found at the 5′ end ofunspliced HIV transcripts, facilitates the transport of unprocessed mRNAfrom the nucleus to the cytoplasm for packaging into virions. Gag isfirst synthesized as a single polypeptide and subsequently cleaved by avirus-encoded protease to yield three structural proteins, p15, p17, andp24. Transdominant inhibitors derived from these gene products have beendemonstrated to inhibit infection of cells cultured with lab pet HIVisolates. One example of a transdominant inhibitor which appears to actby forming nonfunctional multimers with wild-type Rev is RevM10. RevM10construct has blocked infection of CEM cells by HTLV-IIIB for up to 28days (Malim et al. JEM 176:1197, Bevec et al. PNAS 89:9870). In thesestudies, RevM10 failed to demonstrate adverse effect on normal T cellfunction as judged by the criteria of growth rate and IL-2 secretion.

[0166] In another approach T cells can be transduced to producemolecules known as “molecular decoys” which are binding elements forviral proteins critical to replication or assembly, such as TAR. Highlevel retrovirus-mediated expression of TAR in CEM SS cells has beenfound to effectively block the ARV-2 HIV isolate, as measured by RTassay (Sullenger et al. Cell 63:601). Importantly, it also blocked SIV(SIVmac25 1) infection, suggesting that inhibition of HIV infection withmolecular decoys may be generally applicable to various isolates andthereby alleviate the problem of hypervariability. Further, it has beendemonstrated that TAR expression has no discernible effects on cellviability (Sullenger et al. J. Virol. 65:6811). Another “moleculardecoy” which T cells can be transduced to produce is a soluble CD4tagged at the carboxy terminus with a KDEL (lysine-asparticacid-glutamic acid-leucine) sequence, a signal for ER retention(Buonocore and Rose, PNAS 90:2695)(Nature 345:625). The sCD4-KDEL geneexpression is driven by the HIV LTR. H9 cells transduced with thesCD4-KDEL construct show up regulation of expression of intracellularCD4 upon HIV infection. This strategy effectively blocked production ofHIV MN for up to 60 days post infection. The proposed advantage of thisinhibitor is that the virus should not be able to escape it's effect bymutating because CD4 binding is essential for HIV infectivity.

[0167] T cells can also be transduced to express antisense molecules andribozyme which block viral replication or infection. Viral replicationcan be inhibited with a variety of antisense strategies. One particularribozyme which cleaves HIV integrase (Sioud and Drlica, PNAS 88:7303),has been developed and may offer an approach to blocking infection asopposed to merely viral production.

[0168] Another approach to block HIV infection involves transducing Tcells with HIV-regulated toxins. Two examples of this type of approachare the diphtheria toxin A gene (Harrison et al. AIDS Res. Hum. Retro.8:39) and the herpes simplex virus type 1 thymidine kinase gene (HSV TK)(Caruso and Klatzmann, PNAS 89:182). In both cases, transcription wasunder the control of HIV regulatory sequences. While the diphtheriatoxin is itself toxic, the HSV TK requires the addition of acyclovir tokill infected cells. For example the use of HSV TK followed by theaddition of 10 μm acyclovir for 17 days totally blocks HIV infection ofHUT 78 cells for up to 55 days of culture.

[0169] It has been demonstrated that when CD4⁺ T cells from an HIVinfected individual are stimulated with a primary activation signal,such as anti-CD3 antibodies, and anti-CD28 antibodies attached to asolid phase support, such as beads, the cell culture proliferatesexponentially and the amount of HIV particles produced is significantlyreduced as compared to conventional methods for stimulating T cells,such as with PHA and IL-2 (see Examples 16 and 21-27). Thus, when CD4⁺ Tcells from an HIV infected individual are expanded ex vivo with aprimary activation agent, such as an anti-CD3 antibody, and anti-CD28 ona solid phase surface, the presence of anti-retroviral agents may not berequired in the culture to limit replication of HIV. Sinceanti-retroviral drugs have toxic effects on cells, no anti-retroviralagent or reduced amounts of these agents to the T cell culture willresult in expansion to higher cell numbers. Thus, in a preferredembodiment of the invention, CD4⁺ T cells from an HIV infectedindividual are isolated, expanded ex vivo with a primary activationagent and anti-CD28 antibody coated beads (preferably, at a ratio of 3beads per T cell) in the absence of or in the presence of reducedamounts of anti-retroviral agents and readministered to the individual.In an even more preferred embodiment, the primary activation agent is ananti-CD3 antibody, which can be in soluble form or attached to a solidphase support, e.g., the solid phase support on which the anti-CD28antibody is immobilized. Moreover, it has been demonstrated (Example 18)that stimulation of a population of CD4⁺ T cells with anti-CD3 andanti-CD28 antibody coated beads and addition of IL-2 to the cultureresults in polyclonal expansion of the population of T cells in numberof from about 10log₁₀ to 12log₁₀ fold the original CD4⁺ T cellpopulation. Thus, ex vivo expansion of CD4⁺ T cells from an HIV infectedindividual with anti-CD3 and anti-CD28 antibody coated beads and addedIL-2 results in expansion of the CD4⁺ T cells by about 12log₁₀ withsignificantly reduced amounts of virus being produced. The addition oflow doses of anti-retroviral agents to the culture may further limitreplication of HIV without exerting toxic effects on the cells.

[0170] In a further embodiment of the invention, CD4⁺ T cells obtainedfrom an individual and expanded ex vivo according to the method of theinvention can be cryo-preserved. Thus, it is possible to obtain CD4⁺ Tcells from an individual, expand the cells ex vivo, readminister aportion of the expanded population of cells to the individual andcryo-preserve one or several portions of the expanded cell population.This is particularly useful if treatment of the individual requires morethan one administration of CD4⁺ T cells. The cryo-preserved cells canalso be thawed, and expanded according to the method of the invention.Thus, the method of the invention provides a renewable source ofpolyclonal CD4⁺ T cells.

[0171] The invention also provides for in vivo expansion of CD4⁺ T cellsin an individual, particularly in an HIV infected individual. It hasbeen shown that when CD4⁺ T cells infected with HIV are cultured invitro with an agent which provides a primary activation signal, such asan anti-CD3 antibody and anti-CD28 attached to a solid phase surface,expansion of the T cell population is obtained and the amount of HIVproduced is significantly reduced compared to the amount of virusproduced when the cells are stimulated with PHA and IL-2 (Example 15).Moreover, is has been demonstrated (Example 18) that stimulation of apopulation of CD4⁺ T cells with anti-CD3 and anti-CD28 antibody coatedbeads and addition of IL-2 to the culture results in polyclonalexpansion of the population of T cells in number of from about 10log₁₀to 12log₁₀ fold the original CD4⁺ T cell population. Thus, in oneembodiment of the invention, polyclonal expansion of the population ofCD4⁺ T cells in an HIV infected individual is achieved by administrationto the individual of anti-CD3 and anti-CD28 antibodies attached to asolid phase surface, such as biodegradable beads (Bang Laboratory).Additionally, IL-2 can be administered to the individual to furtherpromote CD4⁺ T cell proliferation. This particular embodiment should beuseful as a therapeutic method for increasing the number of CD4⁺ T cellsin an individual, since the expansion of the T cells will occur withlimited HIV replication.

[0172] The the invention also provides methods for restoring theproportion of Th1 versus Th2 cells in an individual having an infection.It has been shown herein (Example 23) that CD4⁺ T cells fromHIV-infected individuals secrete preferentially Th1-type cytokines uponstimulation with immobilized anti-CD28 antibody. Accordingly, treatmentof an HIV-infected individual with immobilized anti-CD28 antibody willresult in a preferential increase of Th1 cells versus Th2 cells. This isparticularly relevant since the ratio of Th1 versus Th2 cells declinesprogressively in HIV-infected patients, which may explain thesusceptibility of these patients to infections by intracellularmicrobes.

[0173] It has been shown herein that expansion of CD4⁺ T cells withimmobilized anti-CD28 antibody results in prevention of infection of theCD4⁺ T cells by HIV-1 (Examples 25-27). It is thus likely that infectionof CD4⁺ T cells by other types of viruses will also be inhibited, or atleast reduced. Infections which can be treated according to the methodsof the invention include infections by viruses that infect CD4⁺ T cells,such as retroviruses. These include oncomaviruses or oncoviruses, suchas human T-lymphotropic virus (HTLV) 1, 2, and 5; and lentiviruses, suchas human immunodeficiency viruses (HIV) 1 and 2. Other types of viruses,such as DNA viruses, including Herpes viruses, e.g., Human HerpesViruses (HHV) and Cytomegalovirus (CMV) and RNA viruses are also withinthe scope of the invention. Accordingly, an individual having a viralinfection, such as a retroviral infection, can be treated in vivo or exvivo by contacting its T cells with an agent which provides acostimulatory signal which inhibits viral production, such as animmobilized anti-CD28 antibody, in the presence of an agent whichdelivers a primary activation signal. The agent which provides a primaryactivation signal can be administered to the individual, or it can be anagent which is already in the individual, such as one or more antigens.The methods of the invention include those that provide treatments ofindividuals infected with a virus that causes a decrease in numbers of Tcells and those that provide treatments of individuals infected with avirus that transforms the T cells, such as HTLV.

[0174] The invention further provides methods for vaccination of anindividual against infection by viruses infecting CD4⁺ T cells and/orother types of cells. In fact, Examples 25-27 demonstrate that CD4⁺ Tcells are protected from infection by HIV when cultured in the presenceof immobilized anti-CD28 antibodies. Accordingly, in one embodiment ofthe invention, a costimulatory agent which blocks or reduces viralproduction, such as immobilized anti-CD28 antibody, is administered toan individual prior to a viral infection. The method can furthercomprise administration to the individual of an agent which provides aprimary activation signal to the T cells.

[0175] Also within the scope of the invention are agents which interactwith CD28 and provide to the T cell a protective effect against a viralinfection. Preferred agents are those which transduce in the T cell asignal significantly similar to that transduced by immobilized anti-CD28antibody, such as the monoclonal antibody 9.3. The invention alsoprovides methods for isolating such agents, by, for example, incubatingT cells with the agent to be tested, adding a virus to the cell culture,and measuring viral production.

[0176] The methods for stimulating and expanding a population of antigenspecific T cells are useful in therapeutic situations where it isdesirable to upregulate an immune response (e.g., induce a response orenhance an existing response) upon administration of the T cells to asubject. For example, the method can be used to enhance a T cellresponse against tumor-associated antigens. Tumor cells from a subjecttypically express tumor-associated antigens but may be unable tostimulate a costimulatory signal in T cells (e.g., because they lacksexpression of costimulatory molecules). Thus, tumor cells can becontacted with T cells from the subject in vitro and antigen specific Tcells expanded according to the method of the invention and the T cellsreturned to the subject. Alternatively, T cells can be stimulated andexpanded as described herein to induce or enhance responsiveness topathogenic agents, such as viruses (e.g., human immunodeficiency virus),bacteria, parasites and fungi.

[0177] The invention further provides methods to selectively expand aspecific subpopulation of T cells from a mixed population of T cells. Inparticular, the invention provides a method to specifically enrich apopulation of CD28⁺ T cells in CD4⁺ T cells. Indeed, stimulation of apopulation of CD28⁺ T cells with anti-CD3 and anti-CD28 antibodies or anatural ligand of CD28, such as B7-1 or B7-2 present on the surface ofCHO cells results in expansion of the population of CD4⁺ T cells at theexpense of the CD8⁺ T cells, which progressively die by apoptosis (seeExample 15). Thus, expansion of CD28⁺ T cells under these conditionsresults in a selective enrichment in CD4⁺ T cells in long term cultures.A population of CD28⁺ T cells can also be stimulated to proliferate andbecome enriched in CD4⁺ T cells by contacting the CD28⁺ T cells with asolid phase surface comprising anti-CD3 and anti-CD28 antibodies or ananti-CD3 antibody and a stimulatory form of B7-2, as described inExample 19.

[0178] Another embodiment of the invention, provides a method forselectively expanding a population of either TH1 or TH2 cells or from apopulation of CD4⁺ T cells. A population of CD4⁺ T cells can be enrichedin either TH1 or TH2 cells by stimulation of the T cells with a firstagent which provides a primary activation signal and a second agentwhich provides a CD28 costimulatory signal i.e., an anti-CD28 antibodyor a natural ligand for CD28, such as B7-1 or B7. For example, toselectively expand TH2 cells from a population of CD4⁺ cells, CD4⁺ Tcells are costimulated with a natural ligand of CD28, such as B7-1 orB7-2, present on the surface of cells, such as CHO cells, to inducesecretion of TH2 specific cytokines, such as IL-4 and IL-5, resulting inselective enrichment of the T cell population in TH2 cells. On thecontrary, to expand TH1 cells from a population of CD4⁺ T cells, CD4⁺ Tcells are costimulated with an anti-CD28 antibody, such as themonoclonal antibody 9.3, inducing secretion of TH1-specific cytokines,including IFN-γ, resulting in enrichment of TH1 cells over TH2 cells(Example 14).

[0179] Compositions and Kits

[0180] This invention also provides compositions and kits comprising anagent which stimulates an accessory molecule on the surface of T cells(e.g., an anti-CD28 antibody) coupled to a solid phase surface and,optionally, including an agent which stimulates a TCR/CD3complex-associated signal in T cells (e.g., an anti-CD3 antibody)coupled to the same solid phase surface. For example, the compositioncan comprise an anti-CD28 antibody and an anti-CD3 antibody coupled tothe same solid phase surface (e.g. bead). Alternatively, the compositioncan include an agent which stimulates an accessory molecule on thesurface of T cells coupled to a first solid phase surface and an agentwhich stimulates a TCR/CD3 complex-associated signal in T cells coupledto a second solid phase surface. For example, the composition caninclude an anti-CD28 antibody coupled to a first bead and an anti-CD3antibody coupled to a second bead. Kits comprising such compositions andinstructions for use are also within the scope of this invention.

[0181] This invention is further illustrated by the following exampleswhich should not be construed as limiting. The contents of allreferences and published patent applications cited throughout thisapplication are hereby incorporated by reference. The followingmethodology described in the Materials and Methods section was usedthroughout the examples set forth below.

[0182] Methods and Materials

[0183] Preparation of Immobilized Anti-CD3 Antibody

[0184] Tissue culture flasks were coated with anti-CD3 monoclonalantibody. Although a number of anti-human CD3 monoclonal antibodies areavailable, OKT3 prepared from hybridoma cells obtained from the AmericanType Culture Collection was used in this procedure. For any anti-CD3antibody the optimal concentration to be coated on tissue culturedflasks must be determined experimentally. With OKT3, the optimalconcentration was determined to be typically in the range of 0.1 to 10micrograms per milliliter. To make coating solution, the antibody wassuspended in 0.05 M tris-HCl, pH 9.0 (Sigma Chemical Co., St. Louis,Mo.). Coating solution sufficient to cover the bottom of a tissueculture flask was added (Falcon, Nunc or Costar) and incubated overnightat 4° C. The flasks were washed three times with phosphate bufferedsaline without calcium or magnesium (PBS w/o Ca or Mg) and blockingbuffer (PBS w/o Ca or Mg plus 5% bovine serum albumin) added to coverthe bottom of the flask and were incubated two hours at roomtemperature. After this incubation, flasks were used directly or frozenfor storage, leaving the blocking solution on the flask.

[0185] Isolation of Peripheral Blood Leukocytes (PBLs)

[0186] Samples were obtained by leukopheresis of healthy donors. Usingsterile conditions, the leukocytes were transferred to a T800 cultureflask. The bag was washed with Hanks balanced salt solution w/o calciumor magnesium (HBSS w/o) (Whittaker Bioproducts, Inc., Walkersville,Md.). The cells were diluted with HBSS w/o and mixed well. The cellswere then split equally between two 200 milliliter conical-bottomsterile plastic tissue culture tubes. Each tube was brought up to 200 mlwith HBSS w/o and spun at 1800 RPM for 12 minutes in a Beckman TJ-6centrifuge. The supernatant was aspirated and each pellet resuspended in50 ml HBSS w/o. The cells were transferred to two 50 ml conical bottomtubes and spun at 1500 RPM for eight minutes. Again the supernatant wasaspirated.

[0187] To lyse the red blood cells, the cell pellets were resuspended in50 ml of ACK lysing buffer (Biofluids, Inc., Rockville Md., Catalog#304) at room temperature with gentle mixing for three minutes. Thecells were again pelleted by spinning at 1500 RPM for 8 minutes. Afteraspirating the supernatant, the pellets were combined into one 50 mltube in 32 ml HBSS w/o.

[0188] Separation of the PBLs from monocytes was accomplished bycentrifugation through a PERCOLL™ gradient. To prepare 1 liter ofPERCOLL™ solution (PERCOLL™-MO), 716 ml of PERCOLL™ (Pharmacia,Piscataway, N.J., Catalog #17-0891-01) was combined with 100 ml 1.5 Msodium chloride, 20 ml 1M sodium-HEPES, and 164 ml water. All reagentsmust be tissue culture grade and sterile filtered. After mixing, thissolution was filtered through a sterile 0.2 μm³ filter and stored at 4°C. 24 ml of PERCOLL™-MO was added to each of two 50 ml conical bottomtubes. To each tube 16 ml of the cell suspension was added. The solutionwas mixed well by gently inverting the tubes. The tubes were spun at2800 RPM for 30 minutes without a brake. The tubes were removed from thecentrifuge, being careful not to mix the layers. The PBLs were at thebottoms of the tubes. Then, the supernatant was aspirated and the PBLswere washed in HBSS w/o by centrifuging for 8 minutes at 1500 RPM.

[0189] Cell Sorting via Negative Magnetic Immunoadherence

[0190] The cell sorting via negative magnetic immunoadherence must beperformed at 4° C. The washed cell pellets obtained from the PERCOLL™gradients described above were resuspended in coating medium (RPMI-1640(BioWhittaker, Walkersville, Md., Catalog #12-167Y), 3% fetal calf serum(FCS) (or 1% human AB- serum or 0.5% bovine serum albumin) 5 mM EDTA(Quality Biological, Inc., Gaithersburg, MD, Catalog # 14-117-1), 2 mML-glutamine (BioWhittaker, Walkersville, Md., Catalog # 17-905C), 20 mMHEPES (BioWhittaker, Walkersville, Md., Catalog # 17-757A), 50 μg/mlgentamicin (BioWhittaker, Walkersville, Md., Catalog #17-905C)) to acell density of 20×10⁶ per ml. A cocktail of monoclonal antibodiesdirected to cell surface markers was added to a final concentration of 1μg/ml for each antibody. The composition of this cocktail is designed toenrich for either CD4⁺ or CD28⁺ T cells. Thus, the cocktail willtypically include antibodies to CD 14, CD20, CD11b, CD16, HLA-DR, and(for CD4⁺ cells only) CD8. (See Table 1 for a list of sorting monoclonalantibody cocktails.) The tube containing cells and antibodies wasrotated at 4° for 30-45 minutes. At the end of this incubation, thecells were washed three times with coating medium to remove unboundantibody. Magnetic beads coated with goat anti-mouse IgG (DynabeadsM-450, Catalog #11006, P&S Biochemicals, Gaithersburg, Md.) andprewashed with coating medium were added at a ratio of three beads percell. The cells and beads were then rotated for 1-1.5 hours at 4° C. Theantibody-coated cells were removed using a magnetic particleconcentrator according to the manufacturer's directions (MPC-1, Catalog#12001, P&S Biochemicals, Gaithersburg, Md.). The nonadherent cells werewashed out of the coating medium and resuspended in an appropriateculture medium. TABLE 1 Sorting Monoclonal Antibody Cocktails:(Italicized mAbs are available from the ATCC) Cocktail TargetsRepresentative mAbs rt-A CD14 63D3 (IgG1), 20.3 (IgM) CD20 1F5(IgG2_(a)), Leu-16 (IgG1) CD16 FC-2.2 (IgG2_(b)), 3G8 (IgG1) HLA-DR 2.06(IgG1), HB10a (IgG) Cocktail Targets Representative mAbs rT-B CD14 63D3(IgG1), 20.3 (IgM) CD21 HB5 (IgG2_(a)) CD16 FC-2.2 (IgG2_(b)), 3G8(IgG1) HLA-DR 2.06 (IgG1), HB10a (IgG) Cocktail Targets RepresentativemAbs r9.3-A CD14 63D3 (IgG1), 20.3 (IgM) CD20 1F5 (IgG2_(a)), Leu-16(IgG1) CD11b OKMI (IgG2_(b)), 60.1 (IgG2_(b)) CD16 FC-2.2 (IgG2_(b)),3G8 (IgG1) HLA-DR 2.06 (IgG1), HB10a (IgG) Cocktail TargetsRepresentative mAbs r9.3-B CD14 63D3 (IgG1), 20.3 (IgM) CD21 HB5(IgG2_(a)) CD11b OKMI (IgG2), 60.1 (IgG2_(b)) CD16 FC-2.2 (IgG2_(b)),3G8 (IgG1) HLA-DR 2.06 (IgG1), HB10a (IgG) Cocktail TargetsRepresentative mAbs rCD4-A CD14 63D3 (IgG1), 20.3 (IgM) CD20 IF5(IgG2_(a)), Leu-16 (IGg1) CD11b OKMI (IgG2_(b)), 60.1 (IgG2_(b)) CD16FC-2.2 (IgG_(b)), 3G8 (IgG1) HLA-DR 2.06 (IgG1), HB10a (IgG) CD8 51.1(IgG2), G10-1.1 (IgG2_(a)), OKT8, (IgG2_(a)) Cocktail TargetsRepresentative mAbs rCD8-B CD14 63D3 (IgG1), 20.3 (IgM) CD20 IF5(IgG2_(a)), Leu-16 (IGg1) CD11b OKMI (IgG2_(b)), 60.1 (IgG2_(b)) CD16FC-2.2 (IgG2_(b)), 3G8 (IgG1) HLA-DR 2.06 (IgG1), HB10a (IgG) CD4G17-2.8 (IgG1) Cocktail Targets Representative mAbs rM0 CD2 35.1(IgG2_(a)), 9.6 (IgG2_(a)) CD20 IF5 (IgG2_(a)), Leu-16 (IGg1) rB CD235.1 (IgG2_(a)), 9.6 (IgG2_(a)) CD14 63D3 (IgG1), 20.3 (IgM) CD11b OKMI(IgG2_(b)), 60.1 (IgG2_(b)) CD16 FC-2.2 (IgG2_(b)), 3G8 (IgG1)

[0191] Long Term Stimulation:

[0192] Tissue culture flasks precoated with anti-CD3 monoclonal antibodywere thawed and washed three times with PBS. The purified T cells wereadded at a density of 2×10⁶/ml. Anti-CD28 monoclonal antibody mAb 9.3(Dr. Jeffery Ledbetter, Bristol Myers Squibb Corporation, Seattle,Wash.) or EX5.3D10, ATCC Deposit No. HB11373 (Repligen Corporation,Cambridge, Mass.) was added at a concentration of 1 μg/ml and cells werecultured at 37° C. overnight. The cells were then detached from theflask by forceful pipetting and transferred to a fresh untreated flaskat a density of 0.5×10⁶/ ml. Thereafter, the cells were resuspendedevery other day by forceful pipetting and diluted to 0.5×10⁶/ ml. Themean diameter of the cells was monitored daily with a Coulter Counter 2Minterfaced to a Coulter Channelyzer. Resting T cells have a meandiameter of 6.8 microns. With this stimulation protocol, the meandiameter increased to over 12 microns by day 4 and then began todecrease by about day 6. When the mean diameter decreased to about 8microns, the cells were again stimulated overnight with anti-CD3 andanti-CD28 as above. It was important that the cells not be allowed toreturn to resting diameter. This cycle was repeated for as long as threemonths. It can be expected that the time between restimulations willprogressively decrease.

EXAMPLE 1 Long Term Growth of CD4⁺ T cells With Anti-CD3 and Anti-CD28Antibodies

[0193] Previous known methods to culture T cells in vitro require theaddition of exogenous feeder cells or cellular growth factors (such asinterleukin 2 or 4) and a source of antigen or mitogenic plant lectin.Peripheral blood CD28⁺ T cells were isolated by negative selection usingmagnetic immunobeads and monoclonal antibodies as described in theMethods and Materials section above. CD4⁺ cells were further isolatedfrom the T cell population by treating the cells with anti-CD8monoclonal antibody and removing the CD8⁺ cells with magneticimmunobeads. Briefly, T cells were obtained from leukopheresis of anormal donor, and purified with FICOLL™ density gradient centrifugation,followed by magnetic immunobead sorting. The resulting CD28⁺, CD4⁺ Tcells were cultured in defined medium (X-Vivo10 containing gentamicinand L-glutamine (Whittaker Bioproducts) at an initial density of2.0×10⁶/ml by adding cells to culture dishes containing plastic-adsorbedGoat anti-mouse IgG (Kirkegaard and Perry Laboratories, Gaithersburg,Md.) and anti-CD3 mAb G19-4. After 48 hours, the cells were removed andplaced in flasks containing either hIL-2 (5%, CalBiochem) or anti-CD28mAb (500 ng/ml). The cells cultured with IL-2 were fed with fresh IL-2at 2-day intervals. Fresh medium was added to all cultures as requiredto maintain a cell density of 0.5×10⁶/ml. Cells were restimulated atapproximately weekly intervals by culture on plastic-adsorbed anti-CD3mAb for 24 hours, the cells removed and placed at 1.0×10⁶/ml in freshmedium in flasks containing either IL-2 or anti-CD28 mAb.

[0194] In the example shown in FIG. 1, the culture vessel initiallycontained 50×10⁶ cells, and the cells were cultured in an optimal amountof mitogenic lectin PHA, or cultured with cyclic stimulation of plasticimmobilized anti-CD3 mAb in the presence of interleukin 2 or anti-CD28mAb 9.3. The cells cultured in PHA alone did not proliferate, with allcells dying by about day 20 of culture, demonstrating the functionalabsence of accessory cells. In contrast, the cells grown in anti-CD3with IL-2 or anti-CD28 entered a logarithmic growth phase, with equalrates of growth for the first three weeks of culture. However, theanti-CD3 cultures began to diverge in growth rates during the fourthweek of culture, with the IL-2 fed cells entering a plateau phase aftera 2.8log₁₀expansion. In contrast, the cultures grown in the presence ofanti-CD28 remained in logarithmic growth until the sixth week ofculture, at which time there had been a 3.8log₁₀ expansion. Thus, CD28receptor stimulation, perhaps by anti-CD28 crosslinking, is able tostimulate the growth of CD4⁺ T cells in the absence of fetal calf serumor accessory cells, and furthermore, about 10-fold more cells can beobtained using anti-CD28 as opposed to addition of exogenous IL-2. Inrepeated experiments, CD4⁺ T cell expansion using anti-CD28 antibodyconsistently yielded more CD4⁺ T cells than expansion using IL-2 (e.g.,up to 1000-fold more cells). This system has the added advantage of notrequiring the presence of accessory cells which may be advantageous inclinical situations where accessory cells are limiting or defective.

EXAMPLE 2 Long Term Growth of Anti-CD28-Treated T cells in MediumContaining Fetal Calf Serum

[0195] Another series of experiments tested whether the growth advantageof CD28 receptor stimulation was due to replacement of factors normallypresent in fetal calf serum. T cells were obtained from leukopheresis ofa normal donor, and purified with FICOLL™ density gradientcentrifugations, followed by magnetic immunobead sorting. The resultingCD28⁺, CD4⁺ T cells were cultured at an initial density of 2.0×10⁶/ml inmedium (RPMI-1640 containing 10% heat-inactivated fetal calf serum[Hyclone, Logan, Utah] and gentamicin and L-glutamine) by adding cellsto culture dishes containing plastic-adsorbed OKT3. After 48 hours, thecells were removed and placed in flasks containing either hIL-2 (10%final concentration, CalBiochem) or anti-CD28 mAb 9.3 (800 ng/ml). Thecells were fed with fresh medium as required to maintain a cell densityof 0.5×10⁶/ml, and restimulated at approximately weekly intervals byculture on plastic adsorbed anti-CD3 mAb for 24 hours.

[0196] As shown in FIG. 2, the cells entered logarithmic growth phase,with equal rates of growth for the first three weeks of culture.However, the anti-CD3 cultures began to diverge in growth rates duringthe fourth week of culture, with the IL-2 fed cells entering a plateauphase after a ¹⁸ 4.0log₁₀expansion. In contrast, the cultures grown inthe presence of anti-CD28 remained in logarithmic growth until the fifthweek of culture, at which time there had been a˜5.1log₁₀ expansion.Thus, CD28 stimulation resulted in a^(˜)125,000-fold expansion of theinitial culture while IL-2 feeding resulted in a 10,000-fold expansionof cells.

EXAMPLE 3 Long Term Growth of T cells in Phorbol Ester, Ionomycin andAnti-CD28-Stimulated T Cells

[0197] Further experiments tested whether alternative methods ofactivating T cells would also permit CD28 stimulated growth.Pharmacologic activation of T cells with PMA and ionomycin is thought tomimic antigen receptor triggering of T cells via the TCR/CD3 complex. Tcells were obtained from leukopheresis of a normal donor, and purifiedwith sequential FICOLL™ and PERCOLL™ density gradient centrifugations,followed by magnetic immunobead sorting. The resulting CD28⁺, CD4⁺ Tcells were cultured at an initial density of 2.0×10⁶/ml by adding cellsto culture dishes containing phorbol myristic acid (PMA 3 ng/ml, Sigma)and ionomycin (120 ng/ml, Calbiochem, lot #3710232). After 24 hours, thecells were diluted to 0.5×10⁶/ml and placed in flasks containing eitherrIL-2 (50 IU/ml, Boerhinger Mannheim, lot #11844900)) or anti-CD28 mAb(1 ug/ml). The cells were fed with fresh medium as required to maintaina cell density of 0.5×10⁶/ml, and restimulated cyclically atapproximately weekly intervals by readdition of PMA and ionomycin. FreshIL-2 was added to the IL-2 containing culture at daily intervals.

[0198] The results of this experiment are shown in FIG. 3. T cells thatwere purified of accessory cells did not grow in cell numbers in thepresence of PMA (“P” in the Figure) and ionomycin (“I” in the Figure),with or without IL-2. The cells clumped and enlarged, as indicated bysize analysis, indicating the cells had been induced to enter the GIphase of the cell cycle but did not progress to DNA synthesis and celldivision. In contrast, addition of CD28 mAb to PMA plus ionomycintreated cells resulted in logarithmic cell growth. Thus, anti-CD3 mAb isnot required to provide T cell activation. It should be appreciated thatother activators of protein kinase C, such as bryostatin ordiacylglycerol can be used in place of PMA.

EXAMPLE 4 Immunophenotype of Cells Cultured with Anti-CD3 Stimulationand Addition of IL-2 or Anti-CD28 mAb

[0199] To examine the subsets of T cells that are expanded, PBL werepropagated for 16 days using either anti-CD3 and IL-2 or anti-CD3 andanti-CD28. FIG. 4 demonstrates the selective enrichment of CD4 cellsfrom peripheral blood lymphocytes. Mononuclear cells were isolated fromblood by ficoll hypaque density gradient centrifugation. The cells werestained with CD4 and CD8 monoclonal antibodies, and analyzed for thepercent positive cells on day 0. The cells were then cultured on plasticimmobilized anti-CD3 monoclonal antibody G19-4 plus IL-2 or plasticimmobilized anti-CD3 monoclonal antibody G19-4 plus anti-CD28 monoclonalantibody 9.3 (0.5 μg/ml). The cells were isolated from culture on day16, and repeat staining for CD4 and CD8 antigens was done by flowcytometry. Data was gated on the lymphocyte population by forward anglelight scatter and side scatter. By this analysis, the % CD4 and CD8cells were 8.0% and 84.5% in the cells grown in IL-2, and 44.6% and52.5% in the cells grown in CD28. These results suggest that CD28expansion favors the CD4⁺ cell, in contrast to the well-establishedobservation that CD8⁺ cells predominate in cells grown in IL-2 (forexample, see D. A. Cantrell and K. A. Smith, (1983), J. Exp. Med.158:1895 and Gullberg, M. and K. A. Smith (1986) J. Exp. Med. 163, 270).

[0200] To further test this possibility, CD4⁺ T cells were enriched to98% purity using negative selection with monoclonal antibodies andmagnetic immunobeads as described above. Fluorescent Activated CellSorter (FACS) Analysis was used to examine the phenotype of the T cellscultured with anti-CD3 and anti-CD28. Cells were pelleted bycentrifugation and resuspended in PBS/1% BSA. The cells were then washedby repeating this procedure twice. The cells were pelleted andresuspended in 100 μl of primary antibody solution, vortexed, and kepton ice for one hour. After washing twice in PBS/1% BSA, the cells wereresuspended in 100 μl of fluorescein-labeled goat-anti-mouse IgG andincubated for 30 minutes on ice. At the end of this incubation, thecells were washed twice in PBS and resuspended in 500 μl 1%paraformaldehyde in PBS. The labeled cells were analyzed on an OrthoCytofluorograph. Cells were stained after isolation, or after 26 days inculture, with phycoerythrin conjugated anti-CD3 (Leu-4), CD4 (Leu-3A),CD8 (OKT8) or with IgG2a control monoclonal antibodies and fluorescencequantified with a flow cytometer. The cells were cultured for one monthusing anti-CD3 and either IL-2 or anti-CD28 to propagate the cells.There was equal expansion of the cells for the first 26 days of theculture (not shown), however, as can be seen in FIG. 5, the phenotype ofthe cells diverged progressively with increasing time in culture so thatat day 26 of culture, the predominant cell in anti-CD28 culture was CD4⁺while the cells in the IL-2 culture were predominantly CD8⁺. Thus, CD28receptor stimulation, perhaps by crosslinking, is able to selectivelyexpand T cells of the CD4 phenotype while the conventional method of invitro T cell culture yields cells of the CD8 phenotype. Additionalexperiments have been conducted with similar results, indicating thatCD28 stimulation of initially mixed populations of cells is able toyield cultures containing predominately or exclusively CD4 T cells, andthus one can expand and “rescue” the CD4 cells that were initiallypresent in limiting amounts.

EXAMPLE 5 Use of Cell Sizing or Cyclic Expression of B7 on CD4⁺ T cellsto Monitor T Cell Expansion

[0201] To determine the time of T cell restimulation, changes in cellvolume were monitored using a Coulter Counter ZM interfaced with aCoulter. CD28⁺, CD4⁺ T cells were isolated as described by magneticimmunoselection, and cultured in the presence of anti-CD28 niAb 9.3 (0.5μg/ml) and restimulated with plastic immobilized anti-CD3 monoclonalantibody G19-4 as indicted. FIG. 9 demonstrates the cyclic changes incell volume during six consecutive restimulations (“S1” to “S6”)performed essentially as described in Example 1. Briefly, cells wereexpanded with anti-CD3 and anti-CD28 over three weeks in culture. Cellswere changed to fresh medium at each restimulation with anti-CD3antibody. Stimulations were spaced at ten day intervals. The cells wererestimulated whenever cell volume decreased to <400 fl.

[0202] In another experiment, cyclic expression of the B7-1 antigen wasused to determine the time for T cell restimulation. The cells obtainedfrom the experiment shown in FIG. 10 were stained with a CTLA-4Ig fusionprotein (obtained from Repligen Corporation; see also Linsley P. S. etal. (1991) J. Exp. Med. 174, 561-569) and analyzed by flow cytometry tomeasure B7-1 receptor expression. It was determined that CD4⁺ T cells donot initially express the B7-1 receptor, and that with culture,expression is induced. Further, the B7-1 expression was found to betransient, and to be re-induced with repeated anti-CD3 restimulation.

EXAMPLE 6 Production of Cytokines by T Cells Following Anti-CD28Stimulation

[0203] Experiments were conducted to analyze the cytokines produced by Tcells following anti-CD28 stimulation. CD28⁺/CD4⁺ T cells were isolatedas described in the previous examples. The cells were stimulated withplastic immobilized anti-CD3 mAb and IL-2 (200 U/ml), or anti-CD3 andanti-CD28 without added lymphokine. The cells were restimulated withanti-CD3 antibody as determined by changes in cell volume as describedin Example 5. Cell culture supernatant was removed at the time pointsindicated and analyzed for IL-2 (FIG. 11), GM-CSF (FIG. 12), and TNF-α(FIG. 13). IL-2 was determined by bioassay on CTLL-2 cells while TNF-αand GM-CSF were measured by ELISA according to manufacturersinstructions (TNFα, GMCSF:R&D Systems, Minneapolis, Minn.). The datashown for the various cytokines are from separate experiments. In otherexperiments (not shown) anti-CD3 plus anti-CD28 stimulation was shown tocause high levels of IL-4 and IL-5 in culture supernatants afterapproximately day 10 of culture, although only small amounts of thesecytokines were present during the early period of culture.

[0204] The patterns of cytokine secretion with cells expanded by severalrestimulations according to the protocol described in the examples wascompared to cells expanded with anti-CD3 plus IL-2 over three weeks inculture. Cells were changed to fresh medium at each restimulation withanti-CD3 antibody. Stimulations were spaced at ten day intervals. After24 hours of further culture, an aliquot of cell culture supernatant wasremoved for assay. ELISA assays for individual cytokines were performedwith kits from various suppliers (IL-2:T Cell Diagnostics, Cambridge,Mass.; IFN-y Endogen, Inc., Boston, Mass.; IL-4, TNFα, GMCSF:R&DSystems, Minneapolis, Minn.) according to directions supplied with thekits. As can be seen from the results of a representative experimentshown in Table 2, the two protocols result in very similar levels ofIL-2 and IL-4 secretion. The higher levels of GM-CSF and TNFA secretionwith anti-CD3 and anti-CD28 costimulation-suggests that theproliferative capacity of this combination of stimuli may be due in partto its ability to stimulate an autocrine loop. TABLE 2 Comparison ofcytokines secreted by T cells expanded with anti-CD3 and IL-2 versus Tcells expanded with anti-CD3 and anti-CD28. Concentration of lymphokinein pg/ml Stimulation Co- cycle stimulus IL-2 IFN-γ IL-4 GM-CSF TNFα S1IL-2 20714  1458  16  2303  789 αCD28 13794  2211  14  3812  3387 S2IL-2 20250 16600  964  51251  3221 αCD28 28411 56600 1030 138207 13448S3 IL-2 21282  8617 1153  86418  2899 αCD28 14129 12583 1044 120418 5969

EXAMPLE 7 Polyclonality of T Cells Following Anti-CD28 Stimulation

[0205] The polyclonality of a population of T cells followingstimulation with an anti-CD3 and an anti-CD28 antibody as described inthe preceding examples was determined. CD28⁺/CD4⁺ T cells were isolatedas described in the previous examples. The cells were stimulated withplastic immobilized anti-CD3 mAb and anti-CD28 mAb and FACS analysisconducted essentially as described in Example 4 using a panel ofanti-TCR antibodies (Vβ5a, Vβ5b, Vβ5c, Vβ6a, Vβ8a, Vβ12a and Vβ2a)obtained from Pharmingen. The polyclonality of the T cell population wasdetermined before (Day 1) and after stimulation (Day 24). As shown inFIG. 14, the TCR diversity of a population of T cells stimulated throughCD28 is maintained at day 24.

EXAMPLE 8 Comparison of Cell Surface Staining of T Cells from HIV⁺ andHIV-Individuals Following Anti-CD28 Stimulation

[0206] Another series of experiments was conducted to determine theexpression of various T cell surface markers on cells from HIVseropositive and seronegative individuals expanded according to theprocedures described in the previous examples. CD28⁺/CD4⁺ T cells wereobtained as described herein. In these experiments, the anti-CD3 mAb waslabeled with a first label (e.g., rhodamine) and the appropriate secondantibody (e.g., anti-CD28, anti-CD4, anti-CD8) was labeled with a secondlabel (e.g., fluorescein). T cells were stimulated with plasticimmobilized anti-CD3 mAb and anti-CD28 mAb as described herein and thepercent of T cells expressing a variety of cell surface markers atdifferenct stimulations (i.e., S1, S2 and S3) determined by FACSanalysis. As shown in FIGS. 15 and 16, the overall cell surface markerdistribution on T cells obtained from HIV seropositive and seronegativeindividuals is approximately the same throughout the stimulation assay.It is noteworthy that the presence of one cell surface marker, CD45RA,which is a marker for naive T cells, declines over the course of CD28stimulated T cell expansion. In contrast, the percent of T cellsexpressing the memory T cell surface marker, CD45RO, increases with CD28stimulation. Thus, T cell expansion through CD28 stimulationpreferentially expands memory T cells or converts naive T cells tomemory T cells. It should be noted that the decline in the percent of Tcells expressing CD28 is an artifact of the experiment due to thepresence of anti-CD28 antibody in the T cell culture throughout theassay. The presence of anti-CD28 antibody prevents staining of the CD28antigen.

EXAMPLE 9 Long Term Growth of CD8⁺ T cells With Anti-CD3 and MonoclonalAntibody 2D8

[0207] Experiments were conducted to determine whether a population ofCD8⁺ T cells could be preferentially expanded by stimulation with ananti-CD3 mAb and a monoclonal antibody 2D8. CD28⁺ T cells were obtainedessentially as described in Example 1. To assay for CD8 expression, aprimary anti-CD8 antibody and a labeled appropriate secondary antibodywere used in FACS analysis to determine the percent positive cells. Asshown in FIG. 17, at day 7 following stimulation of T cells with theanti-CD3 mAb G19-4sp and the mAb 2d8, the CD8⁺ fraction had increasedfrom approximately 20% to over 40%. Another monoclonal antibody ER4.7G11(referred to as 7G11) was also found to stimulate CD8⁺ T cells. Thisantibody was raised against recombinant human CTLA4 and has beendeposited with the ATCC on Jun. 3, 1994 at Accession No. ______. Thisresult indicates that binding of either a distinct region of CTLA4 or ofa cross-reactive cell surface protein selectively activates CD8⁺ Tcells.

EXAMPLE 10 Defining the Epitope of the Monoclonal Antibody 2D8 andCloning the CD9 Antigen

[0208] To detemine the epitope of the monoclonal antibody 2D8, epitopemapping was performed by phage display library (PDL) screening and wasconfirmed using synthetic peptides. A random 20 amino acid PDL wasprepared by cloning a degenerate oligonucleotide into the fUSE5 vector(Scott, J. K. and Smith, G. P. (1990) Science 249:386-390) as describedin Cwirla, S. E. et al. (1990) Proc. Natl. Acad. Sci. USA 87:6378-6382.The PDL was used to identify short peptides that specifically bound mAb2D8 by a micropanning technique described in Jellis, C. L. et al. (1993)Gene 137:63-68. Individual phage clones were purified from the libraryby virtue of their affinity for immobilized mAb and the random peptidewas identified by DNA sequencing. Briefly, mAb 2D8 was coated onto NuncMaxisorp 96 well plates and incubated with 5×10¹⁰ phage representing8×10⁶ different phage displaying random 20 amino acid peptides.Specifically bound phage were eluted, amplified, then incubated with theantibody a second time. After the third round, 7 phage were isolated,and DNA was prepared for sequencing.

[0209] Sequence analysis of these clones demonstrated that three of theseven sequences were identical and a fourth was similar:

[0210] 2D8#2(SEQ ID NO: 8) H Q F C D H W G C W L L R E T H I F T P 2D8#4

[0211] (SEQ ID NO:8) H Q F C D H W G C W L L R E T H I F T P

[0212] 2D8#10(SEQ ID NO: 8) H Q F C D H W G C W L L R E T H I F T P

[0213] 2D8#6 (SEQ ID NO: 9) L R L V L E D P G I W L R P D Y F F P A

[0214] Based on this data an epitope of G X W L X D/E (SEQ ID NO: 12)was proposed.

[0215] In addition to CTLA4, a second antigen for mAb 2D8 was discoveredusing cDNA expression cloning.

[0216] A. Construction of a cDNA Expression Library

[0217] A cDNA library was constructed in the pCDM8 vector (Seed, (1987)Nature 329:840) using poly (A)+RNA isolated from activated T cells asdescribed (Aruffo et al. (1987) Proc. Natl. Acad. Sci. USA 84:3365). Toprepare total RNA, T cells were harvested from culture and the cellpellet homogenized in a solution of 4 M guanidine thiocyanate, 0.5%sarkosyl, 25 mM EDTA, pH 7.5, 0.13% Sigma anti-foam A, and 0.7%mercaptoethanol. RNA was purified from the homogenate by centrifugationfor 24 hour at 32,000 rpm through a solution of 5.7 M CsCl, 10 mM EDTA,25 mM Na acetate, pH 7. The pellet of RNA was dissolved in 5% sarkosyl,1 mM EDTA, 10 mM Tris, pH 7.5 and extracted with two volumes of 50%phenol, 49% chloroform, 1% isoamyl alcohol. RNA was ethanol precipitatedtwice. Poly (A)⁺ RNA used in cDNA library construction was purified bytwo cycles of oligo (dT)-cellulose selection.

[0218] Complementary DNA was synthesized from 5.5 μg of poly(A)⁺ RNA ina reaction containing 50 mM Tris, pH 8.3, 75 mM KCl, 3 mM MgCl₂, 10 mMdithiothreitol, 500 μM dATP, dCTP, dGTP, dTTP, 50 μg/ml oligo(dT) 12-18,180 units/ml RNasin, and 10,000 units/ml Moloney-MLV reversetranscriptase in a total volume of 55 μl at 37° C. for 1 hr. Followingreverse transcription, the cDNA was converted to double-stranded DNA byadjusting the solution to 25 mM Tris, pH 8.3, 100 mM KCl, 5 mM MgCl2,250 μM each dATP, dCTP, dGTP, dTTP, 5 mM dithiothreitol, 250 units/mlDNA polymerase I, 8.5 units/ml ribonuclease H and incubating at 16° C.for 2 hr. EDTA was added to 18 mM and the solution was extracted with anequal volume of 50% phenol, 49% chloroform, 1% isoamyl alcohol. DNA wasprecipitated with two volumes of ethanol in the presence of 2.5 Mammonium acetate and with 4 micrograms of linear polyacrylamide ascarrier. In addition, cDNA was synthesized from 4 μg of poly(A)⁺ RNA ina reaction containing 50 mM Tris, pH 8.8, 50 μg/ml oligo(dT)₁₂₋₁₈, 327units/ml RNasin, and 952 units/ml AMV reverse transcriptase in a totalvolume of 100 μl at 42° C. for 0.67 hr. Following reverse transcription,the reverse transcriptase was inactivated by heating at 70° C. for 10min. The cDNA was converted to double-stranded DNA by adding 320 μl H₂Oand 80 μl of a solution of 0.1M Tris, pH 7.5, 25 mM MgCl₂, 0.5 M KCl,250 μg/ml bovine serum albumin, and 50 mM dithiothreitol, and adjustingthe solution to 200 μM each dATP, dCTP, dGTP, dTTP, 50 units/ml DNApolymerase I, 8 units/ml ribonuclease H and incubating at 16° C. for 2hours. EDTA was added to 18 mM and the solution was extracted with anequal volume of 50% phenol, 49% chloroform, 1% isoamyl alcohol. DNA wasprecipitated with two volumes of ethanol in the presence of 2.5 Mammonium acetate and with 4 micrograms of linear polyacrylamide ascarrier.

[0219] The DNA from 4 μg of AMV reverse transcription and 2.0 μg ofMoloney MLV reverse transcription were combined. Non-selfcomplementaryBstXI adaptors were added to the DNA as follows: The double-strandedcDNA from 6 μg of poly(A)⁺ RNA was incubated with 3.6 μg of a kinasedoligonucleotide of the sequence CTTTAGAGCACA (SEQ ID NO: 13) and 2.4 μgof a kinased oligonucleotide of the sequence CTCTAAAG (SEQ ID NO: 14) ina solution containing 6 mM Tris, pH 7.5, 6 mM MgCl₂, 5 mM NaCl, 350μg/ml bovine serum albumin, 7 mM mercaptoethanol, 0.1 mM ATP, 2 mMdithiothreitol, 1 mM spermidine, and 600 units T4 DNA ligase in a totalvolume of 0.45 ml at 15° C. for 16 hours. EDTA was added to 34 mM andthe solution was extracted with an equal volume of 50% phenol, 49%chloroform, 1% isoamyl alcohol. DNA was precipitated with two volumes ofethanol in the presence of 2.5 M ammonium acetate.

[0220] DNA larger than 600 bp was selected as follows: The adaptored DNAwas redissolved in 10 mM Tris, pH 8, 1 mM EDTA, 600 mM NaCl, 0.1%sarkosyl and chromatographed on a Sepharose CL-4B column in the samebuffer. DNA in the void volume of the column (containing DNA greaterthan 600 bp) was pooled and ethanol precipitated.

[0221] The pCDM8 vector was prepared for cDNA cloning by digestion withBstXI and purification on an agarose gel. Adaptored cDNA from 6 μg ofpoly(A)⁺ RNA was ligated to 2.25 μg of BstXI cut pCDM8 in a solutioncontaining 6 mM Tris, pH 7.5, 6 mM MgCl₂, 5 mM NaCl, 350 μg/ml bovineserum albumin, 7 mM mercaptoethanol, 0.1 mM ATP, 2 mM dithiothreitol, 1mM spermidine, and 600 units T4 DNA ligase in a total volume of 1.5 mlat 15° C. for 24 hr. The ligation reaction mixture was then transformedinto competent E.coli DH10B/P3 by standard techniques. Plasmid DNA wasprepared from a 500 ml culture of the original transformation of thecDNA library. Plasmid DNA was purified by the alkaline lysis procedurefollowed by twice banding in CsCl equilibrium gradients (Maniatis et al,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y.(1987)).

[0222] B. Cloning Procedure

[0223] In the cloning procedure, the cDNA expression library wasintroduced into MOP8 cells (ATCC No. CRL1709) using lipofectamine andthe cells screened with mAb 2D8 to identify transfectants expressing a2D8 ligand on their surface. In the first round of screening, thirty 100mm dishes of 50% confluent COS cells were transfected with 0.05 μg/mlactivated T cell library DNA using the DEAE-Dextran method (Seed, B. etal. (1987) Proc. Natl. Acad. Sci. USA 84:3365). The cells weretrypsinized and re-plated after 24 hours. After 47 hours, the cells weredetached by incubation in PBS/0.5 mM EDTA, pH 7.4/0.02% Na azide at 37°C. for 30 min.

[0224] Detached cells were treated with 10 μg/ml mAb 2D8. Cells wereincubated with the monoclonal antibody for 45 minutes at 4° C. Cellswere washed and distributed into panning dishes coated withaffinity-purified goat anti-mouse IgG antibody and allowed to attach atroom temperature. After 3 hours, the plates were gently washed twicewith PBS/0.5 mM EDTA, pH 7.4/0.02% Na azide, 5% FCS and once with 0.15 MNaCl, 0.01 M Hepes, pH 7.4, 5% FCS. Unbound cells were thus removed andepisomal DNA was recovered from the adherent panned cells byconventional techniques.

[0225] Episomal DNA was transformed into E. coli DH10B/P3. The plasmidDNA was re-introduced into MOP8 cells using lipofectamine and the cycleof expression and panning was repeated twice. Cells expressing a 2D8ligand were selected by panning on dishes coated with goat anti-mouseIgG antibody. After the third round of screening, plasmid DNA wasprepared from individual colonies and transfected into MOP8 cells by theDEAE-Dextran method. Expression of a 2D8 ligand on transfected MOP8cells was analyzed by indirect immunofluorescence with mAb 2D8 (See FIG.18).

[0226] DNA from one clone (mp5) identified as positive by FACS analysiswas sequenced using standard techniques. FASTA analysis of the aminoacid sequence of mp5 identified a matching protein, CD9, in the GCG databanks. The full amino acid sequence of CD9 is shown below (SEQ ID NO:6).

[0227] BESTFIT analysis of the phage epitopes of mAb 2D8 to the aminoacid sequence of CD9 revealed a close match: G C W L L R E (phage 2D8#2,4,10; SEQ ID NO:10) G I W L R P D (phage 2D8#6; SEQ ID NO:11) G L W L RF D (CD9 sequence; SEQ ID NO:7)

[0228] FT DOMAIN 111 194 EXTRACELLULAR (PROBABLE) FT TRANSMEM 195 220POTENTIAL FT DOMAIN 221 227 CYTOPLASMIC (PROBABLE) FT CARBOHYD 51 51POTENTIAL FT CARBOHYD 52 52 POTENTIAL FT CONFLICT 8 8 C→S(IN REF.1) FTCONFLICT 66 66 G→A(IN REF.1) FT CONFLICT 193 193 MISSING (IN REF.1) SQSEQUENCE 227 AA; 25285 MW; 261251 CN;

[0229] Cd9_Human Length: 227 May 25, 1994 14:10 Type: P Check: 1577 (SEQID NO: 6)   1 PVKGGTKCIK YLLFGFNFIF WLAGIAVLAI GLWLRFDSQT KSIFEQETNN  51NNSSFYTGVY ILIGAGALMM LVGFLGCCGA VQESQCMLGL FFGFLLVIFA 101 IEIAAAIWGYSHKDEVIKEV QEFYKDTYNK LKTKDEPQRE TLKAIHYALN 151 CCGLAGGVEQ FISDICPKKDVLETFTVKSC PDAIKEVFDN KFHIIGAVGI 201 GIAVVMIFGM IFSMILCCAI RRNREMV

EXAMPLE 11 Induction of T Cell Expansion by Costimulation with B7-1 orB7-2

[0230] In order to determine whether costimulation through CD28 can beprovided by B7-1 and B7-2 molecules expressed on cells transfected witha nucleic acid encoding either of these molecules, Chinese Hamster Ovary(CHO) cells were transfected with human B7-1 (CD80) or B7-2 (CD 86)(Freedman, A. S. et al. (1987) J. Immunol. 137:3260-3267; Freeman, G. J.et al. (1989) J. Immunol. 143:2714-2722; Freeman, G. J. et al. (1991) J.Exp. Med. 174:625-631; Freeman, G. J. et al. (1993) Science 262:909-911;Azuma, M. et al. (1993) Nature 366:76-79; Freeman, G. J. et al. (1993)J. Exp. Med. 178:2185-2192). The cells were maintained in G418 asdescribed in Gimmi et al.(1991) PNAS 88:6575 and Engel et al. (1994)Blood 84:1402. Briefly, a cDNA fragment of B7-1 containing nucleotides86-1213 (comprising the coding region) was inserted into the eukaryoticexpresssion vector pLEN (Metabolic Biosystem, Mountain View, Calif.)containing the human metallothionein IIA promoter, the simian virus 40enhancer, and the human growth hormone 3′ untranslanted region andpolyadenylation site. A cDNA fragment of B7-2 containing the codingregion was inserted into pLEN. Fifty micrograms of Pvu I linearizedB7-pLEN construct was cotransfected with 5 micrograms of linearizedSV2-Neo-SP65 into CHO cells by electroporation using the BRLelectroporator at settings of 250 V and 1600 μF. Transfectants wereselected by growth in medium containing the neomycin analogue G418sulfate (400 μg/ml) and were cloned. Mock transfected CHO cells weremade by transfection of linearized SV2-Neo-Sp65 alone.

[0231] To determine the relative expression of human B7-1 and B7-2 onthe stably transfected CHO cells, the cells were stained with CTLA4Ig(obtained from Repligen Corporation) and FITC conjugated goat anti-humanIgG Fc, or with anti-B7-1 monoclonal antibody 133 (Freeman et al. J.Immunol. 137:3260 (1987)) and FITC goat anti-mouse IgM, or with anti-B70(B7-2) monoclonal antibody IT2 (obtained from Pharmingen Corporation)and FITC goat anti-mouse IgG, fixed in paraformaldehyde and fluorescenceanalyzed by flow cytometry. The B7-1-CHO cells expressed about twice asmany binding sites than the B7-2-CHO cells for CTLA4Ig. Control CHOcells (CHO-neo) consisted of CHO cells transfected with the neomycinresistance vector, and did not stain specifically for CTLA4Ig.Specificity of ligand expression was confirmed by measurement withanti-CD80 mAb BBI (Yokochi, T. et al. (1982) J. Immunol. 128:823-827)and anti-CD86 mAb IT2. Mitomycin C treatment did not affect B7expression.

[0232] In order to explore whether costimulation by the CD28 and CTLA4ligands B7-1 and B7-2 is similar, CD28⁺ T cells were cultured withanti-CD3 in the presence of B7-1 or B7-2-transfected CHO cells, orcontrol CHO-neo cells (FIG. 19). For this experiment, CD28⁺ T wereisolated from peripheral blood lymphocytes by Percoll gradientcentrifugation from leukopacks obtained by apheresis of healthy donorsand purified by negative selection according to June et al. (1987) Mol.Cell Biol. 7:4472. Purified T cells were cultured in RPMI 1640containing 10% heat-inactivated fetal calf serum (Hyclone, Logan UT), 2mM L-glutamine, and 20 mM Hepes in 96 well flat bottom microtiter platesat 37° C. in 5% CO₂. The CD28⁺ T cells (5×10⁴ cells/well) werestimulated with anti-CD3 monoclonal antibody coated beads (3 beads per Tcell) in the presence of mitomycin C-inactivated CHO cells expressingB7-1, B7-2, or neomycine resistance gene only (2×10⁴ cells/well). Cellswere also stimulated with anti-CD3 monoclonal antibody OKT3 coated beadsin the presence of anti-CD28 mAb 9.3 at 1 μg/ml. In preliminaryexperiments, the anti-CD28 mAb was titered to determine the optimalamounts for induction of IL-2 secretion. The anti-CD3 monoclonalantibody OKT3 (IgG2a), obtained from the ATCC, was bound to magneticbeads (M-450, Dynal Corp.) that were coated with goat anti-mouse IgG byadding 150 femtograms of antibody per bead, and the beads washedextensively. In all experiments, antigen presenting cells were firstremoved from the CD28⁺ T cells by immunomagnetic bead depletion. CHOcells were inactivated by pretreatment with mitomycin C at 25 μg/ml forone hour. On days 2 to 4 of culture, thymidine incorporation experimentswere performed by pulsing the cultures overnight withmethyl-³H-thymidine (New England Neclear) with 37 kBq/well andincorporation determined by liquid scintillation spectroscopy. Resultswere expressed as the mean ±SEM cpm of triplicate determinations.

[0233] The results of the experiment are shown graphically in FIG. 19.On day 3 of culture, low level proliferation was observed in cellsstimulated with anti-CD3 in the presence of control CHO cells, andlevels of thymidine incorporation did not increase with further culture.In contrast, there was increasing thymidine incorporation in T cellsthat were co-cultured with CHO cells that express B7-1 or B7-2 that wasdetected after 2 to 4 days of culture, and this cellular proliferationcontinued until exhaustion of culture medium. Levels of thymidineincorporation in B7-1 and B7-2 stimulated cultures was similar tocultures stimulated with anti-CD3 plus anti-CD28 monoclonal antibody.Thus, B7-1 and B7-2 molecules expressed on CHO cells can costimulate Tcells as efficiently as anti-CD28 monoclonal antibody.

[0234] In preliminary experiments, culture of T cells with B7-1 or B7-2expressing CHO cells alone or in combination did not result in T cellproliferation, consistent with previous reports that B7-1 alone is notmitogenic (Liu et al. (1992) Eur. J. Immunol. 22:2855). Anti-CD28stimulation in combination with PMA has previously been shown to becomitogenic. To determine whether B7-1 or B7-2 were comitogenic withPMA, purified CD28⁺ T cells were cultured in PMA (10 nM) with mitomycinC-inactivated CHO cells expressing B7-1, B7-2, a 1:1 mixture of B7-1plus B7-2 expressing CHO cells, or control CHO-neo cells only, or withanti-CD28 monoclonal antibody (1 μg/ml), and the time course of cellularproliferation determined. The results are shown graphically in FIG. 20.Proliferation was less vigorous in the presence of 10 nM PMA than whencells were cultured with anti-CD3 mAb (FIG. 20). The time course ofproliferation was similar in cultures that contained B7-1, B7-2 or amixture of cells expressing B7-1 plus B7-2. In contrast, cells culturedin PMA only or PMA plus CHO-neo cells incorporated low amounts ofthymidine, consistent with the rigorous removal of antigen presentingcells. Thus, B7-1 or B7-2 molecules expressed on CHO cells can provide acostimulatory signal to T cells activated either by anti-CD3 mAb or PMA,but proliferation is stronger with anti-CD3 mAb. Resting T cells do notexpress detectable amounts of CTLA-4, however activated T cells expressboth CD28 and CTLA-4 by day 2 of culture (Linsey et al.(1992) J. Exp.Med. 176:1595). Given that both B7-1 and B7-2 bind to CD28 and CTLA-4,it was possible that some component of cellular activation could beattributed to CTLA-4, compatible with previous studies indicating thatCTLA-4 ligation can enhance comitogenic effects of suboptimal CD28ligation (Damle et al. (1994) J. Immunol. 152:2686). For thisexperiment, anti-CD28 Fab fragments were produced by papain digestion of9.3 mAb and two cycles of purification on a protein A column (Pierce,Rockford, Ill.). CD28⁺ T cells were cultured in the presence of anti-CD3coated beads and CHO (as described above) cells for three days in thepresence of increasing amounts of CD28 Fab fragments. Cells were pulsedwith tritiated thymidine and scintillation counting performed on theindicated day of culture. IL-2 concentration in culture supernatants wasdetermined by ELISA using a commercially available kit from T CellDiagnostics (Cambridge, Mass.) after 24 hours of culture. The valuesreported were assessed by using dilutions of culture supernatant thatyielded read-outs within the linear portion of the standard curve.

[0235] The results are shown graphically in FIG. 21. Proliferationinduced by both B7-1 and B7-2 was inhibited >95% in a dose-dependentmanner by CD28 Fab fragments, indicating that both forms of B7costimulation are critically dependent on interaction with CD28. IL-2accumulation in culture supernatants was also efficiently blocked by theFab fragments. Together these results demonstrate that both B7-1 andB7-2 molecules expressed on CHO cells are capable of costimulating Tcell proliferation similarly to costimulating T cell proliferation withCD28 mAbs.

EXAMPLE 12 Costimulation with B7-1 and B7-2 Induce Long TermProliferation of CD4⁺ T Cells

[0236] The ability of the B7 ligands to sustain T cell proliferation inlong term cultures was investigated. For these experiments, CD4⁺ T cellswere obtained from CD28⁺ T cells by negative selection using magneticbeads (Dynal) coated with CD8 monoclonal antibodies as described in Juneet al. (1989) J. Immunol. 143:153. The phenotype of the cells was 99%CD2⁺, 98% CD28⁺, and 96% CD4⁺. 5×10⁶ purified CD4⁺ T cells werestimulated with anti-CD3 monoclonal antibody coated beads (1.5 10⁷beads) and mitomycin C-inactivated CHO cells (2.10⁶ cells) expressingB7-1, B7-2, or neomycin resistance only, or with anti-CD3 plus anti-CD28coated “cis” beads, i.e. with both antibodies on the same bead. Beadswere coated with anti-CD3 (OKT3) and anti-CD28 9.3 monoclonal antibodywith each antibody added at 150 femtograms per bead. It is important tonote that no cytokines were added to the culture medium so that cellgrowth was dependent on secretion of cytokines and lymphokines. Freshmedium was added at two to three days intervals with fresh medium tomaintain cell concentrations between 0.5-1.5×10⁶ T cells/ml;antibody-coated beads and CHO cells were not cleared from culture, butwere diluted progressively until restimulation. The cell cultures weremonitored by electronic cell sizing using a Coulter Counter model ZM andChannelyzer model 256 (Coulter, Hialeah, Fla.), and restimulated atapproximately 7 to 10 day intervals (i.e. when the volume of the T cellblasts decreased to <400 fl) with additional beads and mitomycinC-treated CHO cells. Viable T cells were counted and the total number ofcells that would be expected to accumulate displayed, taking intoaccount discarded cells.

[0237] The results are shown graphically in FIG. 22. Both B7-1 and B7-2resulted in exponential T cell expansion, and during the first threeweeks of culture both B7 receptors could consistently induce a ≧3 logloof expansion that was polyclonal. Together, the above results indicatethat either B7-1 or B7-2 can costimulate long-term proliferation in aCD28-dependent manner, independent of a requirement for simultaneousB7-1 plus B7-2 receptor coexpression. To date, no phenotypic differenceshave been detected in the cells that arise from B7-1 or B7-2 stimulatedCD4⁺ T cell expansion. T cells cultured with anti-CD3 and control CHOcells did not undergo sustained proliferation, and the culture wasterminated due to poor viability. Thus, B7-1 and B7-2 expressed on CHOcells induced long term T cell proliferation of activated CD4⁺ T cellssimilarly to costimulation with anti-CD28 mAbs.

EXAMPLE 13 Differences in Cytokines Secreted from B7 versus Anti-CD28stimulated T cells in Short Term T Cell Cultures

[0238] The experiments described in Examples 11 and 12 did not revealany notable differences between B7-1, B7-2, or anti-CD28 mAb in theability to provide a costimulatory signal for the induction ormaintenance of T cell proliferation. To further test whether anti-CD28or B7-1 and B7-2 mediated distinct costimulatory effects, theaccumulation of various cytokines was examined in T cell subsets. Tofirst assess global effects of B7 costimulation on T cells, CD28⁺ Tcells were stimulated with plastic immobilized anti-CD3 mAb and titeredamounts of B7-1 or B7-2 CHO transfectants or anti-CD28 mAb 9.3 at 1μg/ml. Anti-CD3 mAb (OKT3) was precoated on the culture wells byovernight incubation with a 10 μg/ml solution. Supernatants of the Tcell cultures were collected after 24 h of culture and analyzed by ELISAusing commercially available kits. The kits were obtained from thefollowing sources: IL-2, T Cell Diagnostics, Cambridge, MA; TNF-alpha,GM-CSF, IL-4, and IL-5, R&D Systems, Minneapolis, Minn.;Interferon-gamma, Endogen, Boston, Mass. All values were assessed byusing dilutions of culture supernatant that yielded read-outs within thelinear portion of the standard curve. The cytokine production data issummarized below in Table 3.

[0239] No cytokines were detected in supernatants from cells cultured inmedia alone, as would be expected with resting T cells. Similarly,anti-CD3 did not elicit detectable amounts of IL-2, although low levelsof IFNγ, TNF-α and GM-CSF were present after anti-CD3 stimulation alone.Addition of either B7-1 or B7-2 CHO cells resulted in a dose dependentincrease in cytokines. The costimulatory effect was most marked in thecase of IL-2, as both B7-1 and B7-2 resulted in 1 00-fold or moreaugmentation of IL-2 secretion in comparison to CD3 plus control CHOcell cultures. There was also a dose-dependent increase in IFN-γ, IL-4,TNF-αand GM-CSF secretion induced by B7-1 and B7-2. Importantly, B7-1and B7-2 costimulation elicited nearly equivalent amounts of all testedcytokines. There were however some differences between the amount ofcytokines secreted by cells stimulated with anti-CD28 mAb as compared toB7-1 or B7-2; most notably, both B7-1 and B7-2 were associated withhigher levels of IL-4 secretion (B7-1, 200; B7-2, 250; anti CD28, <20pg/ml). TABLE 3 Effects of B7-1 and B7-2 on Lymphokine Production byCD28⁺ T Cells. (cytokine concentration in pg/ml) Culture Condition (CHOcell/T cell ratio) IL-2 IFN-γ IL-4 TNFa GM-CSF Medium <60 <10 <20 <30<100 αCD3⁺CHO-B7-1(0.1) 1790 2640 62 1250 2490 αCD3⁺CHO-B7-1(0.2) 22503880 110 1690 3110 αCD3⁺CHO-B7-1(0.4) 4040 4430 149 1910 3260αCD3⁺CHO-B7-1(0.8) 5500 5930 200 2110 3670 αCD3⁺CHO-B7-2(0.1) 1620 347070 1310 2990 αCD3⁺CHO-B7-2(0.2) 3640 5490 145 2040 4040αCD3⁺CHO-B7-2(0.4) 5826 7640 220 2440 4420 αCD3⁺CHO-B7-2(0.8) 6830 8610250 2410 4260 αCD3⁺CHO-neo(0.1) <60 480 <20 670 520 αCD3⁺CHO-neo(0.2)<60 650 <20 690 570 αCD3⁺CHO-neo(0.4) <60 690 <20 745 800αCD3⁺CHO-neo(0.8) <60 690 <20 605 725 αCD3 only <60 280 <20 380 280αCD3⁺CD28 Mab 9.3 1630 860 <20 1760 2090

[0240] As shown in Table 3, B7-1 and B7-2-induced cytokine accumulationappeared to plateau at similar ratios of CHO cells to T cells (about 0.4CHO cells: T cell), suggesting that a failure to detect differencesbetween B7 receptors was not due to a differential dose response betweenB7-1 and B7-2. Further, this indicates that neither B7 receptor had beentested under limiting conditions. However, it was possible thatdifferential effects of B7-1 and B7-2 exist, and that this would beapparent only in T cell subsets. Alternatively, it was possible thatintrinsic differences between B7-1 or B7-2 would only be revealed afterrepeated T cell costimulation, to permit possible cellulardifferentiation.

[0241] To assess whether distinct T cell subsets might differentiallyrespond to B7-1 or B7-2,CD28⁺ T cells were divided into CD28⁺CD4⁺ andCD28⁺CD8⁺cells and into CD4⁺CD45RO⁺ and CD4⁺CD45RA+subsets. To obtainCD28⁺CD4⁺ or CD28⁺CD8⁺ T cells, the CD28⁺ T cells were subjected to asecond round of negative selection using magnetic beads (Dynal) coatedwith CD8 or CD4 mAb as described in June et al. (1989) J. Immunol.143:153. CD4⁺CD45RO⁺ and CD4⁺CD45RA⁺ subpopulations were isolated bysubjecting CD28⁺ T cells to negative selection using magnetic beads andanti-CD45RO monoclonal antibody UCHL1 or anti-CD45RA monoclonal antibodyALB11 (Immuntech).

[0242] To examine cytokine production, cells were stimulated withplastic-immobilized anti-CD3 Abs and CHO-B7-1 or CHO-B7-2 cells or withplastic beads expressing both anti-CD3 and anti-CD28 (as describedabove). The results are summarized below in Table 4. Table 4 shows thatB7-1 and B7-2 elicited similar amounts of IL-2 secretion from CD4⁺ Tcells. Both receptors stimulated CD8⁺ T cells with equal efficiency,however about 4-fold less IL-2 accumulated in the supernatants from CD8⁺T cells. Anti-CD28 caused potent costimulation of IFNγ in both CD4⁺ andCD8⁺ T cell subsets. B7 receptors could also elicit high levels of IFN-γfrom either subset, although the magnitude of the effect was two tofour-fold less than anti-CD28 mAb stimulation. TABLE 4 Effects of B7-1and B7-2 on lymphokine production by CD4⁺ and CD8⁺ T cells (cytokineconcentrations in pg/ml) Culture Condition IL-2 IFN-γ GM-CSF CD4⁺ Cellsmedium <60 <15 <15 αCD3 93 1270 9770 αCD3⁺CD28 mAbs 87500 44200 77300αCD3⁺CHO-neo 60 1760 9380 αCD3⁺CHO-B7-1 5320 14200 32400 αCD3⁺CHO-B7-23940 18300 30100 CD8⁺ Cells medium <60 <15 <15 αCD3 133 1620 8900αCD3⁺CD28 mAbs 53600 26200 69600 αCD3⁺CHO-neo 100 3530 8830αCD3⁺CHO-B7-1 1360 10700 20600 αCD3⁺CHO-B7-2 1320 14600 20700

[0243] It was surprising that CD8⁺CD28⁺ T cell cultures accumulatedsimilar amounts of IL-2, IFN-γ and GM-CSF as did CD4⁺CD28⁺cells. TheCD8⁺ subpopulation contained <2% CD4⁺ cells, and thus contamination ofthe CD8⁺ cells by CD4⁺ cells can not explain the nearly equivalentlevels of cytokine secreted by the CD4 and CD8 subsets. In theexperiment shown in Table 4, cells were stimulated with beads expressingboth anti-CD3 and anti-CD28 mAbs (“cis” stimulation), while in theexperiment shown in Table 3, plastic-immobilized anti-CD3 plus fluidphase anti-CD28 (“trans”) costimulation was used. “Cis” stimulation wasconsistently more efficient at eliciting cytokine accumulation (Table 4vs. Table 3, and see FIGS. 25 and 26 below).

[0244] The costimulatory signals provided by anti-CD28, B7-1 and B7-2were similarly potent in eliciting accumulation of GM-CSF (Tables 3 and4). This is most apparent if the fold elevation above anti-CD3 mAb only,or control CHO cultures is examined when comparing cultures stimulatedwith anti-CD28 or B7-1 and B7-2. With regard to TNFα, both B7-1 and B7-2had similar costimulatory effects on CD4⁺ T cells.

[0245] While the effects of B7-1 and B7-2 appeared quite similar on CD4⁺and CD8⁺ T cell subsets, it remained possible that distinct functionsmight be revealed in CD4 subsets. Purified CD4⁺ T cells weremagnetically sorted into “virgin” CD45RA⁺ and “memory” CD45RO⁺populations as described above. The cells were then tested for theirability to secrete IL-2, IL-4 and IFNγ after stimulation with anti-CD3mAb coated beads in the presence of B7-1 or B7-2-expressing CHO orcontrol neo-CHO cells (see FIGS. 23 and 24). The experiment in FIG. 23was carried out in RIO medium and the experiment of FIG. 24 was carriedout in Aim V medium. Supernatants were harvested after 24 hours ofculture, and cytokine concentrations were measured by ELISA as describedabove. Both B7-1 and B7-2 were able to costimulate both subsets toequivalent levels of proliferation. However, striking differences wereuncovered in the induction of cytokine secretion by B7 from CD45RO⁺ andCD45RA⁺ subpopulations. B7-1 and B7-2 costimulation resulted in thesecretion of large amounts of IL-2 from both subsets (FIG. 23, left handpanels). In contrast, neither B7-1 nor B7-2 could elicit IL-4 (FIG. 23and 24, right hand panels) or IFNγ (FIG. 24, left hand panels) from theCD45RA⁺ subpopulation. Since both B7-1 and B7-2 were tested at a varietyof T cell to CHO cell ratios, ensuring that responses were assessed atplateau levels of costimulation, it is unlikely that either receptor hasa differential costimulatory function on CD4⁺ T cells with naive andmemory phenotypes. Furthermore, no differential sensitivity or primingof these cellular subsets to low levels of B7 stimulation was observed;B7-1 and B7-2 mediated lymphokine responses occurred at similarthresholds in both naive and memory subsets.

EXAMPLE 14 Differential Cytokine Secretion upon Costimulation withAnti-CD28 in “cis”, in “trans”, or with B7 molecules in Long TermCulture

[0246] The experiments described in Examples 11, 12 and 13 indicate thatB7 receptors have similar costimulatory effects on the cytokinesproduced during the first round of T cell activation and division, andindicate that CD4 subpopulations have differential capacity to secretecytokines. In this example, differences in the ability of B7-1 or B7-2to induce differentiation were investigated. CD4⁺ CD28⁺ T cells werestimulated with anti-CD3 in the presence of CHO cells expressing B7-1 orB7-2, or with anti-CD3 plus CD28 mabs on beads in “cis” as described inExample 12. The cells were maintained in exponential growth during theexperiment. Supernatants were collected after 24 hours of culture and onday 11, cells were washed and placed in fresh medium, restimulated withfresh anti-CD3 and CHO cells, and supernatants collected after a further24 hours culture. Cytokine levels were measured as described above.

[0247] The results are shown graphically in FIGS. 25-27. During theinitial 24 hours of activation, B7-1 and B7-2 induced nearly equivalentamounts of IL-4, and this was more than that induced by anti-CD28 (FIG.25, bottom panels). In contrast, costimulation with B7-1 and B7-2 didnot elicit as much IL-2 as anti-CD28 stimulation during the first 24hours (FIG. 25, top). These results are consistent with Table 4, whereanti-CD28 stimulation in “cis” was also employed. However, onrestimulation of cells with B7 receptors on day 12 of culture, about10-fold more IL-4 accumulated when compared to the initial stimulationof resting T cells. The anti-CD28 mAb mediated increase in IL-4secretion on restimulation was not as striking as with B7 restimulation.In contrast, anti-CD28 was more efficient than B7 in the induction ofIL-2 secretion during restimulation.

[0248] IL-5 secretion was not detectable after primary stimulation withanti-CD3 and anti-CD28, while both B7-1 and B7-2 resulted in low-levelIL-5 secretion of similar magnitude during the first day of stimulation(FIG. 25, bottom). A notable increase in B7-1 and B7-2-mediated IL-5secretion occurred on day 12 restimulation. IL-5 secretion alsoincreased after anti-CD28 restimulation, however, the increase was about8 to 10-fold less than that due to B7 restimulation. Thus, the patternof IL-5 secretion is similar to that of IL-4 (FIG. 25 vs. 26, bottompanels).

[0249] The effects of restimulation on IFNγ were also examined. Highlevel IFNγ secretion occurred within 24 hours after B7 and anti-CD28primary stimulation (FIG. 26, top), consistent with Table 4. Atrestimulation, however, anti-CD28 was superior to both B7 receptors.Thus, the pattern of anti-CD28 and B7-mediated IFNγ secretion is similarto that of IL-2, and the pattern of IL-5 secretion is similar to that ofIL-4 (FIG. 25 vs. 26). FIG. 27 shows the effects of anti-CD28 and B7restimulation on GM-CSF and TNFα secretion by CD4⁺ T cells. With regardsto GM-CSF, both anti-CD28 and B7-1 and B7-2 increased GM-CSF secretion,although the fold costimulation was more modest, at 4 to 8-fold overthat induced by anti-CD3 plus control CHO cells (FIG. 27, bottom). Onrestimulation, there were no consistent differences between the variousCD28 ligands in the ability to promote GM-CSF secretion. In contrast,anti-CD28 was more effective than B7 receptors at maintaining TNFαsecretion on restimulation (FIG. 27, top). Thus, during the initialactivation of T cells, and during reactivation of CD4⁺ T cell blasts invitro, no consistent differences between B7-1 and B7-2 could beidentified in any of the cytokines examined. Interestingly however,anti-CD28 mAb favored IL-2, IFNγ, and TNFα secretion, while B7-1 andB7-2 promoted IL-4 and IL-5 secretion. Thus, costimulation with B7-1 orB7-2 resulted in preferential secretion of TH2-specific cytokines,whereas costimulation with anti-CD28 resulted in preferential secretionof TH1-specific cytokines. The above results did not reveal anyconsistent differences in the induction of cytokine secretion by B7-1and B7-2 while differences between anti-CD28 and B7 were observed inFIGS. 25-26 and in Table 3. To determine a potential mechanism for thesedifferences, CD28⁺ T cells were cultured in the presence of anti-CD3plus anti-CD28 beads in “cis” (both antibodies on the same bead) or in“trans” (both antibodies on different beads). For “cis” stimulation,immunomagnetic beads were coated with OKT3 and 9.3 mAbs with eachantibody added at 150 femtograms per bead and added at a ratio of 3beads per T cell. For “trans” stimulation, an equal amount anti-CD3 andanti-CD28 coated beads were added at a ratio of 3 (of each type) beadsper T cell. In addition, cells were cultured with anti-CD3 plusanti-CD28 beads in “cis” with mitomycin-inactivated CHO-neo cells addedat a ratio of 2.5:1 T cell to CHO cell, as a control for factorsintrinsic to CHO cells. Finally, T cells were cultured with anti-CD3beads and CHO-B7-1 cells at a ratio of 2.5:1 T cell to CHO cell. Thecells were cultured as indicated in Example 12.

[0250] The results of these experiments are shown graphically in FIGS.28-29. All conditions induced exponential expansion of CD4 T cells (FIG.29). However, there were differences in the cytokines induced by theseforms of costimulation (FIG. 28). Cells stimulated with anti-CD28 in“cis” or with anti-CD28 in “cis” plus CHO-neo cells maintained highlevels of IL-2 secretion. In contrast, anti-CD28 stimulation in “trans”was less efficient at inducing IL-2 secretion, and this form ofcostimulation was progressively less efficient upon repetitiverestimulation. The anti-CD3 plus CHO-B7-l stimulation in “trans” was theonly condition that resulted in progressively increasing amounts of IL-4secretion, consistent with the results shown in FIG. 26 and Table 3.Together, the above results demonstrate that B7-1 and B7-2 both have theability to stimulate T cell proliferation and cytokine secretion andthat the manner of CD28 costimulation can have substantial effects onthe patterns of cytokine secretion.

EXAMPLE 15 Cell Death by Apoptosis of CD8⁺ T Cells Costimulated withAnti-CD28

[0251] Previous information in this application shown in FIG. 4 hasshown that CD28 costimulation can favor the growth of CD4⁺ T cells. Todetermine a mechanism for this effect, CD8⁺ T cells were isolated bynegative immunomagnetic selection. The cells were stimulated withanti-CD3 antibody coupled to beads (b) or to a solid phase (SP) i.e.,tissue culture dish plus anti-CD28 in “cis” or in “trans”. The inductionof apoptosis was assessed by detecting single strand DNA breaks using aflow cytometric TdT assay (Gorczyca, W., J. Gong, and Z. Darzynkiewicz1993. Detection of DNA strand breaks in individual apoptotic cells bythe in situ terminal deoxynucleotidyl transferase and nick translationassays. Cancer Res. 53:1945). During the first 48 hours of culture (S1),the cells became activated and <10% of cells underwent apoptosis (Table5). The cells were restimulated twice with anti-CD3 and anti-CD28 whenrequired and the induction of apoptosis again assesed 24 to 48 hoursfollowing each restimulation (S2 and S3). A dramatic increase in DNAbreaks was uncovered. This was not prevented with the addition ofrecombinant IL-2 (rIL-2) at 100 U/ml. TABLE 5 Apoptosis assays of CD8⁺ Tcells costimulated with anti-CD28 % TdT Positive by Flow CytometryStimulation T0 T24 hr T48 hr S1 OKT3/9.3 3b/c 2 6 1 OKT3 3b/c + 9.3 3b/c2 3 1 OKT3 3b/c + soluble 9.3 2 3 1 OKT3sp + soluble 9.3 2 1 2 Medium 22 2 S2 OKT3/9.33b/c + rhIL2 40 15 39 OKT3 3b/c + 9.3 3b/c 22 47 63 OKT33b/c 9.3 3b/c + rhIL2 22 55 34 OKT3 3b/c + soluble 9.3 27 70 72 OKT33b/c + soluble 9.3 + rhIL2 27 76 59 S3 OKT3/9.3 3b/c 67 OKT3/9.3 3b/c +rhIL2 11 75 OKT3 3b/c + 9.3 3b/c + rhIL2 19 52

[0252] It was next determined if the induction of apoptosis in CD8⁺ Tcells was specific to the CD8⁺ T cell subset. CD4⁺ and CD8⁺ T cells wereisolated and stimulated separately with anti-CD3 and anti-CD28antibodies. During the first 48 hours of culture (SI), there was littleevidence of programmed cell death in either the CD4 or the CD8⁺ T cellsubsets. In contrast, on restimulation (S2), there was a markedinduction of cell death in the CD8⁺ T cell subset (Table 6). Again, thiswas not prevented by addition of exogenous IL-2. Thus, the selectiveinduction of CD8⁺ T cell death is one mechanism that permits CD28stimulation to enhance CD4⁺ T cell expansion. The absence of programmedcell death in the CD4⁺ T cells is consistent with the observations shownelsewhere in this application that the CD4⁺ cells remain polyclonal forextended periods of culture. TABLE 6 Apoptosis assays of CD4 and CD8⁺ Tcells % TdT Positive by Flow Cytometry CD4 and CD8 T cells T0 T24 hr T48hr S1 CD4 OKT3/9.3 3b/c (cis) 5 10 9 CD4 OKT3 3b/c + rhIL2 5 9 9 CD4OKT3 3b/c + 9.3 3b/c (trans) 5 12 11 CD8 OKT3/9.3 3b/c (cis) 3 6 3 CD8OKT3 3b/c + rhIL2 3 6 12 CD8 OKT3 3b/c + 9.3 3b/c (trans) 3 7 9 S2 CD4OKT3/9.3 3b/c (cis) 6 18 13 CD4 OKT3 3b/c + rhIL2 4 27 9 CD4 OKT3 3b/c +9.3 3b/c (trans) 6 13 15 CD8 OKT3/9.3 3b/c (cis) 8 63 47 CD8 OKT3 3b/c +rhIL2 15 75 62 CD8 OKT3 3b/c + 9.3 3b/c (trans) 20 79 61

[0253] The selective cell death of CD8⁺ T cells and not CD4⁺ T cellswill be useful in selectively enriching a population of T cells, forexample CD28⁺ T cells in CD4⁺ T cells during expansion.

EXAMPLE 16 Expansion of CD4⁺ T Cells from HIV Infected Individuals

[0254] In order to determine whether CD4⁺ T cells from individualsinfected with HIV can similarly be expanded ex vivo, CD4⁺ T cells wereobtained from HIV infected individuals and activated with anti-CD3 andanti-CD28 coated beads (3 beads containing an equimolar amount of eachantibody, per T cell) in the presence or absence of anti-retroviraldrugs. CD4⁺ T cells from a patient with 430 CD4⁺ T cells/μl wereisolated by negative selection as described in Example 12 and incubatedin standard RPMI 10% fetal calf serum either in the presence or absenceof the following anti-retroviral drugs: AZT (available fromBurroughs-Wellcome) at 1 μM, DDI (available from Bristol Myers Squibb)at 10 μM, and Nevirapine (available from Boehringer Mannheim) at 1 μM.The growth curves of the cells are represented in FIG. 30. The T cellsexpanded exponentially by a factor of more than 10,000 fold either inthe presence or absence of added anti-retroviral drugs. Moreover, theCD4⁺ T cells expanded to higher numbers in the absence of the drug. Inaddition, no significant amounts of HIV-1 was detected in eitherculture, as the amount HIVp24 present in the supernatant of the cultures(determined by the Spearmen-Karber method) was <50 pg/ml. TheSpearmen-Karber method is described in Richman D. B., Johnson V. A.,Mayrs V. L. 1993, In vitro evaluation of experimental agents foranti-HIV activity. Current Protocols in Immunology ch. 12.9, Colligan J.E., Kruisback A. M., Margolis D. H., Shevach E. M., Strober W. Ed.Greene and Wiley. Interscience NY.

[0255] To further investigate the finding that the amount of HIVproduced in the CD4⁺ T cell cultures stimulated in the presence ofanti-CD3 and anti-CD28 was very low, the extent of replication of thevirus was compared directly between CD4⁺ T cells isolated from HIV (US 1isolate) infected cells (according to Richman D. B., Johnson V. A.,Mayrs V. L. 1993, In vitro evaluation of experimental agents foranti-HIV activity. Current Protocols in Immunology ch. 12.9, Colligan J.E., Kruisback A. M., Margolis D. H., Shevach E. M., Strober W. Ed.Greene and Wiley. Interscience NY), stimulated with PHA (5 μg/ml) andIL-2 (100 U/ml) or with anti-CD3 and anti-CD28. The TCID₅₀ wasdetermined by the method of Spearmen-Karber at days 7, 14, and 21 of thecultures. As shown in Table 7, there was a marked difference between thetiter of virus in the supernatants of T cells stimulated with anti-CD3and anti-CD28 coated beads (3 beads per T cell) as compared to theconventional method of propagation using PHA and IL-2. The mechanism forthis interesting effect is not yet known, but it suggests anotherpotential mechanism for “rescuing” uninfected CD4 cells in cultures fromHIV seropositive patients. TABLE 7 CD4⁺ T Cells stimulated by anti-CD3and anti-CD28 do not support replication of HIV-1. TCID₅₀ CellStimulation Day 7 Day 14 Day 21 anti-CD3 + anti-CD28 <64 <64 <64 PHA +IL-2 18820 >65000 >65000

[0256] It was possible that CD28 causes the proliferation of aparticular subset of lymphocytes that are resistant to infection byHIV-1. Alternatively, it was possible that the mechanism of stimulationper se was able to confer resistance or sensitivity to HIVinfection/expression. To test these possibilities, PBMC were obtainedfrom a normal blood donor, and either the purified CD4⁺ T (105cells/well) cells or whole PBMC (1 05 cells/well) activated with PHA (5μg/ml) or with anti-CD3 and anti-CD28 coated beads (3 beads per T cell).The cells were infected with a T cell trophic variant of HIV-1 (US1) ora monocyte trophic variant (BAL) on day 2 of culture as described inRichman D. B., Johnson V. A., Mayrs V. L. 1993, In vitro evaluation ofexperimental agents for anti-HIV activity. Current Protocols inImmunology ch. 12.9, Colligan J. E., Kruisback A. M., Margolis D. H.,Shevach E. M., Strober W. Ed. Greene and Wiley. Interscience NY. Thelevel of virus expression was quantitated in the culture supernatants onday 7 as shown in Table 8 by the Spearmen-Karber method. In PBMC, highlevels of virus were expressed if the cells were stimulated with PHAwhereas very low or no levels of virus were detected in culturesstimulated with anti-CD3 and anti-CD28 antibodies. This result wasobtained whether or not plastic adherent monocytes/macrophages (M/M)were added to the culture (10⁴ cells per well). TABLE 8 Virus titrationsin PHA and CD3/CD28-stimulated PBMCs with or without addition ofmonocytes/macrophages. US1 TCID50 BAL TCID50 Cell Group Day 7 Day 7PBMC + PHA 332555 241029 PBMC + anti-CD3/CD28 279 77 CD4 cells + M/M +PHA >390625 >390625 CD4 cells + M/M + anti- 386 279 CD3/CD28 CD4 cellsanti- 1012 386 CD3/CD28

[0257] Thus, stimulation of CD4⁺ T cells infected with HIV with anti-CD3and anti-CD28 results in much lower amounts of HIV particles produced ascompared to the conventional method of T cell stimulation with PHA andIL-2. Moreover, since exponential growth of the cells was observed forat least 40 days, this method could therapeutically be useful for exvivo and in vivo expansion of CD4⁺ T cells from an individual infectedwith HIV.

EXAMPLE 17 Large Scale Expansion of CD4⁺ T Cells using Anti-CD3 andAnti-CD28 Antibodies

[0258] To determine if the small scale expansion of T cells withanti-CD3 and anti-CD28 antibodies is also functional on a larger scale,required for clinical use, CD4⁺ T cells were obtained from a normaldonor and cultured in either 3 liter gas-permeable bags (Baxter) or inT75 flasks (FIG. 31). The large scale culture system was seeded with5×10⁷ cells. The cells were stimulated with a bead:cell ratio of 3:1 andthe beads contained an equimolar amount of anti-CD3 (OKT3) and anti-CD28(mAb 9.3) antibodies. The cells cultured in T75 were restimulated at day12 (S2) wit anti-CD3 and anti-CD28 coated beads in the same conditionsas for the first stimulation. The cells cultured in the gas-permeablebags were restimulated at day 11 (S2). No exogenous cytokines were addedto the large scale culture system. The T75 flasks were carried out induplicate, one containing exogenous IL-2 (100 μ/ml) and the other withno added cytokine. The large scale culture grew equivalent to the smallscale culture system, and was harvested on day 19 of culture and 2.4×1010 CD4⁺ T cells recovered. Viability was >95%. Thus, CD4⁺ T cells can beexpanded to high cell numbers in large cultures by stimulating the Tcells with anti-CD3 and anti-CD28 coated beads, and will thus be usefulfor clinical uses.

EXAMPLE 18 10log₁₀ to 12log₁₀ Polyclonal Expansion of CD4⁺ T Cells

[0259] Primary cells exhibit in vitro growth curves characterized by alag phase, an exponential growth phase, and a plateau phase. In FIGS.1-3 and 22 of this application, CD4⁺ T lymphocytes were shown to enterthe plateau phase of growth after 41 og 10 to 5 log 10 expansion. Thefollowing modifications to the culture system enable 10log₁₀ to 12log₁₀polyclonal expansion of CD4⁺ T cells. The modifications consist of usingchemically immobilized anti-CD28 antibody or CD28 natural ligand to thesurface to the tissue culture vessel or beads. This minimizes orprevents the loss of CD28 receptor on the surface of the expandingcells. In addition, IL-2 is added to the culture medium when the cellsbegin to produce limiting amounts of IL-2.

[0260] In the experiment shown in FIG. 32, anti-CD3 and CD28 coatedbeads were prepared by tonsyl conjugation according to themanufacturer's instructions (tosyl activated Dynal M450 beads) asdescribed in example 14. Approximately 5×10⁶ CD4⁺ T cells obtained bynegative selection as described in previous Examples were cultured inthe presence of the antibody coated beads at a ratio of 3 beads per Tcells. The anti-CD3/CD28 coated beads were maintained in the cellculture, and added as needed when cell volume decreased to <400fl, asdescribed earlier in this application. The cultures were performed inthe absence of exogenous cytokines or feeder cells as described earlierin the application. At day 49 the culture was divided in two, andcontinued as before or continued with the addition of rIL-2 (100 U/ml).

[0261]FIG. 32 indicates that the cells continued to divide for a totalof 10.6log10 expansion using this modified protocol where IL-2 wasmaintained by addition at alternate day intervals. The cells in thisculture retained a phenotype consistent with the original CD4⁺ T cellsand remained polyclonal, as judged by T cell receptor VB chain surfaceexpression (FIG. 33) determined as described in Example 7 using a panelof anti-TCR antibodies.

[0262] The results of several experiments are summarized in Table 9. Inthe first 9 experiments CD4⁺ T cells were cultured using anti-CD3 andCD28 antibody coated beads in the absence of exogenous added cytokinesor feeder cells, according to the protocol described in Example 1. In 6of the cultures, the experiments were continued until the cells reacheda plateau stage of growth, and the magnitude of expansion in theseexperiments ranged from 4.0log₁₀ to 6.9log₁₀. In terms of populationdoublings, this represents 14 to 23 cell divisions. In two experiments(Exps# NC020795 and NPW1215), CD4⁺ T cells cells were cultured usinganti-CD3 and anti-CD28 coated beads and IL-2 added to supplement themedium when the growth rate first began to slow down. As seen in thesetwo experiments, a 10.6log₁₀ to 12log₁₀ expansion was achieved,representing 35 to 40 population doublings. The mean population doublingtime has ranged from 30 to 59 hours, and this does not appear to bealtered by the modifications described above. The growth rates of thecell cultures were subjected to linear regression analysis, and allcurves were modeled by exponential growth with regression coefficientsof >0.97 (Table 9), consistent with the polyclonal cell division in theabsence of cell death. TABLE 9 CD4⁺ T Cell Expansion in the presence orabsence of added IL-2 Fold PD Exp # Cells Day 0 Day end expansion # daysStop Point Medium Time (hr) r2 leu10-6 CD4 5.0E+06 3.5E+12 7.08E+05 41plateau R10 30.5 0.993 It32GC CD4 2.0E+07 2.1E+11 1.06E+04 31 plateauR10 54.0 0.998 022395c CD4 5.0E+06 1.5E+11 2.92E+04 28 still linear R1043.9 0.997 30995D CD4 4.0E+06 3.1E+09 7.75E+02 15 still linear R10 34.00.995 271ta CD4 2.5E+07 2.6E+10 1.03E+03 24 still linear R10 47.3 0.9931t13 CD4 2.5E+07 4.9E+12 1.96E+05 50 plateau R10 59.7 0.967 Tr5a CD45.0E+07 7.9E+11 1.57E+04 26 plateau R10 37.2 0.990 NPW 10-13 CD4 3.5E+063.2E+13 9.03E+06 40 plateau R10 37.1 0.990 NPW1111 CD4 5.0E+06 6.2E+121.23E+06 48 platear R10 55.1 0.976 NC020795 CD4 6.0E+06 2.7E+17 4.56E+1078 plateau R10/IL-2 48.6 0.997 NPW1215 CD4 2.0E+06 2.0E+18 1.00E+12 78plateau R10/IL-2 53.8 0.972

[0263] Thus, culture of CD4⁺ T cells in the presence of anti-CD3 andanti-CD28 antibody coated beads and the addition of IL-2 when cellgrowth slows down results in the expansion of the population of T cellsby about 10log₁₀ to 12log₁₀, far in excess of that currentlydemonstrated in the art. This method of culture of CD4⁺ T cells hasutility for clinical use both for in vitro cell culture and genetransduction and for in vivo uses. Furthermore, this demonstrates thefinding that cells produced as described earlier in this application andshown in FIGS. 1-3 which reached a plateau phase of growth after 4log₁₀to 6log₁₀ expansion actually have an extended inherent replicativepotential.

EXAMPLE 19 B7-2Ig Fusion Protein can Provide Sufficient Costimulation toMaintain Long Term Growth of CD4⁺ T Cells

[0264] Example 18 demonstrated that long term proliferation of CD4⁺ Tcells can be obtained by stimulating the cells with anti-CD3 andanti-CD28 antibody coated beads. The experiments described in Example 12showed that long term proliferation of CD4⁺ T cells can be achieved whenthe cells are costimulated with B7-1 or B7-2 expressing CHO cells. Theexperiments described in this Example indicate that long termproliferation of CD4⁺ T cells can be obtained by stimulating the cellswith anti-CD3 antibody and B7-2Ig coated beads, but not with anti-CD3antibody and B7-lIg coated beads.

[0265] Soluble fusion proteins of human B7-1 and B7-2 consisting of theextracellular domain of B7-1 and B7-2 and the Fc portion of human IgG1were prepared as described earlier in the application. These fusionproteins were coupled to tosyl-activated Dynal beads in the presence ofequimolar amounts of OKT3 monoclonal antibody. The adhesion function ofthe B7-1 and B7-2 fusion proteins was assesscd by their ability to bindto Jurkat cells that have the cell surface phenotype: CD28⁺ CTLA4⁻. Bothfusion proteins were shown to bind specifically to CD28 on Jurkat cellsby immunofluorescence analysis.

[0266] CD28⁺ T cells were divided into the CD4 and CD8⁺ T cell subsetsas described in Example 13. The cell subsets were stimulated with beadscoated with either OKT3 alone, or with OKT3 and B7-1Ig, B7-2Ig, oranti-CD28 antibody 9.3. No cytokine or feeder cells were added to thesecultures, and the cultures were maintained as described in Example 1.

[0267] The results are presented in FIG. 34. The results indicate that,consistent with the data shown in Examples 15 and 18, anti-CD28 antibodymaintained long term growth of CD4⁺ T cells, with the CD4⁺ T cellsentering the plateau stage of growth on day 25 of culture after 3.5log₁₀expansion. In contrast, CD8⁺ T cells entered a plateau phase of growthafter 17 days of culture and a 2log₁₀ expansion. Surprisingly, neitherthe CD4 nor CD8⁺ T cells subset was induced to expand substantially withOKT3/B7-1 with both cultures entering plateau phase by day 10 ofculture. In contrast, OKT3/B7-2 was most potent as CD4⁺ T cells expandedfor 30 days. The cells stimulated with beads coated with OKT3 only diedwithin 1 week of culture, confirming that the starting population of CD4and CD8⁺ T cells was in fact, depleted of accessory cells that mighthave provided costimulation through endogenous B7 receptors.

[0268] These results indicate that the natural ligand B7-2 is useful forthe selective expansion of CD4⁺ T cells, and confirm earlier findingsthat anti-CD28 antibody is useful for selective expansion of CD4⁺ Tcells. In contrast, the surprising failure of B7-1 to maintain growth ofCD4 or CD8⁺ T cells indicates that this receptor has a distinct functionand is not sufficient for full costimulation.

EXAMPLE 20 Determination of the Amount of IL-2 Required for OptimalProliferation of CD4⁺ T Cells

[0269] It has been demonstrated in Example 18 that stimulation of CD4⁺ Tcells with anti-CD3 and anti-CD28 antibody coated beads in the presenceof added IL-2 results in polyclonal expansion of the CD412log₁₀population of about 12log₁₀ the number of T cells in the originalpopulation. This example describes a method that can be used todetermine the optimal amount of IL-2 that should be added to the T cellculture to obtain optimal proliferation of the cells.

[0270] CD4⁺ T cells are isolated by negative selection as previouslydescribed. 5×10⁶ purified CD4⁺ T cells are stimulated with anti-CD3 andanti-CD28 monoclonal antibody coated beads (1.5 10⁷ beads). Beads arecoated with anti-CD3 (OKT3) and anti-CD28 9.3 monoclonal antibody witheach antibody added at 150 femtograms per bead. No cytokines are addedto the culture medium so that cell growth was dependent on secretion ofcytokines and lymphokines. Fresh medium was added at two to three daysintervals with fresh medium to maintain cell concentrations between0.5-1.5×10⁶ T cells/ml; antibody-coated beads are not cleared fromculture, but are diluted progressively until restimulation. The cellcultures are monitored by electronic cell sizing using a Coulter Countermodel ZM and Channelyzer model 256 (Coulter, Hialeah, Fla.), andrestimulated at approximately 7 to 10 day intervals (i.e. when thevolume of the T cell blasts decreases to <400 fl) with additional beads.

[0271] The amount of IL-2 present in the supernatant is measured everysecond day, before new medium is added to the culture. The amount ofIL-2 is determined by ELISA as described above. The number of T cells inthe culture is determined on the same days as the IL-2 concentration.The amount of IL-2 and the cell number are then represented graphically.The range of concentrations of IL-2 that is optimal for CD4⁺ T cellsproliferation corresponds to the concentration of IL-2 present in thesupernatant of the culture at the time the growth curve is steapest.

[0272] Thus, knowing the preferred amount of IL-2 that is required foroptimal T cell proliferation, determined as described in this Example,it is possible to supplement a culture of T cells with IL-2 to obtainfinal levels of IL-2 of about the preferred amount.

EXAMPLE 21 Expansion of CD4⁺ T cells from HIV-infected Individuals withLimited Viral Burden

[0273] This experiment demonstrates that stimulation of CD4⁺ T cellsfrom HIV-infected individuals with anti-CD3 antibody and anti-CD28antibody attached to beads results in exponential growth of the cellsfor at least 50 days and reduced viral production as compared tostimulation of the cells with PHA and IL-2.

[0274] CD4⁺ T cells from patients with intermediate-stage HIV-1infection were isolated by negative selection as described herein andcultured in plastic tissue culture flasks at 1-2×10⁶ cells per ml inRPMI 1640 containing 10% heat-inactivated fetal calf serum (Hyclone,Logan Utah), 2 mM L-glutamine, and 20 mM HEPES. Cells were culturedeither in PHA 5 μg/ml and rIL-2 at 100 U/ml (Boehringer Mannheim) orwith anti-CD3 OKT3 plus anti-CD28 9.3 mAbs coated immunomagnetic beads(Dynal) that were loaded with equal amounts of each type of antibody.Beads were added at 3 beads per T cell. The cell cultures were monitoredby electronic cell sizing and restimulated with additional beads whenthe volume of the T cell blasts decreased to <400 fl. No exogenouscytokines or feeder cells were added to the culture. Cells andsupernatants were harvested at 5 to 7 days intervals for analysis ofcell growth and viral expression.

[0275] Viral expression was measured by 3 criteria: (a) the amount ofp24 in the supernatant, as determined by the Spearmen-Karber methoddescribed herein; (b) the amount of gag DNA in the cells; and (c) theamount of gag RNA in the cells. The amount of gag DNA and RNA wasdetermined by a quantitative polymerase chain reaction. Accordingly,frozen cell pellets containing 2×10⁶ cells were resuspended in 200 μllysis buffer [10 mM Tris-HCl, (pH7.5), 2.5 mM MgCl₂, 0.45% Triton X-100(Boehringer Mannheim), 0.45% Tween 20 (Biorad), and 0.12 mg/mlproteinase K (Boehringer Mannheim)]. HIV gag RNA sequences were reversetranscribed into DNA according to techniques known in the art. HIV-1 gagDNA sequences were amplified as described (Vahey et al. PCR Primer: ALaboratory Manual. Dieffenbach and Dveksler, Eds. (Cold Spring HarborLaboratory Press, 1995), p. 17 and p. 313; Vahey et al. J. Virol. 66,310 (1992)). The amplified products were detected by liquidhybridization with end-labeled oligonucleotide probes, followed by gelelectrophoresis. PCR products were quantitated as described (Vahey etal., supra) using a Molecular Dynamics phosphorimager. Standardizationof the PCR products was achieved by parallel amplification of a seriesof plasmid external control templates.

[0276] The results are represented in FIG. 35, Panels A-D. Panel A,depicting growth curves of CD4⁺ T cells from an HIV-infected individual,shows that in the PHA-stimulated culture, the growth curve revealed aninitial exponential expansion and a subsequent plateau phase resultingin termination on day 18 of the culture. This was coincident withincreasing p24 antigen production and increased viral burden, asmeasured by quantitative PCR for cellular HIV-1 gag (Panels B-D). Incontrast, when cells were cultured with anti-CD3 and anti-CD28 antibody,exponential cell proliferation was maintained for at least 50 days(Panel A). While there was evidence of modest viral expression early inthe culture as indicated by p24 levels on day 8 of culture (Panel B),viral production and proviral DNA decreased to undetectable levels inthe culture (Panels C and D). Similar results were obtained whether thestarting cell population was peripheral blood mononuclear cells orpurified CD4⁺ T cells, indicating that the enhanced cell proliferationand antiviral effects in the culture stimulated with anti-CD3 and CD28were CD8⁺ T cell independent and not accessory-cell dependent. Thus,culture of CD4⁺ T cells from HIV-infected individuals with anti-CD3 andanti-CD28 coated beads results in exponential growth of the cells withonly limited viral production.

[0277] Next, CD4⁺ T cells from 10 HIV-infected individuals (CDC category2A or 2 B infections) were cultured in the presence ofanti-CD3/anti-CD28 coated beads as described above in this example. Thecells from three of these patients, Patients 1-3, were further treatedwith combinations of the antiretroviral drugs AZT at 1 μM, DDI at 5 μM,and Nevirapine at 0.5 μM, as indicated in Table 10, while the cells fromPatients 4-10 were cultured without drugs. The percentage of CD4⁺ Tcells was determined at the beginning and at the end of the culture andthe expansion of the culture determined. Viral production was determinedat 7 to 14 day intervals in the cultures by measure of p24 levels inculture supernatants and measure of the amount of gag RNA and DNA in thecells. TABLE 10 Anti-CD3/anti-CD28 mediated expansion of CD4⁺ T cellsfrom HIV-infected individuals Log CD4 Cell Initial Final CD4 Ct CultureCD4 (%) Expansion^(a) p24^(c) Gag RNA Gag RNA Patient (/mm³) TreatmentInitial Final (Days) (ng/ml) (copies/10⁵ cells) (copies/10⁵ cells) 1 554A, D 93.2 97.1 3.7 (35) <0.1 55 <3 2 433 A, D 93.2 95.8 2.8 (29) <0.1224 <3 3 430 A, D, N 93.4 97.2 4.0 (34) <0.1 7090 15 4 445 None 92.398.7 3.1 (28) <0.1 147 23 5 355 None n.d.^(b) 95.8 4.5 (40) <0.1 8 <3 6384 None 91.7 39 3.3 (36) <0.1 313 <3 7 466 None 93.6 95.8 3.6 (28) <0.164 <3 8 500 None 82.8 67.7 2.2 (28) <0.1 267 <3 9 401 None n.d. 97.8 6.9(71) 0.17 2448 <10 10 413 None 64.1 70.2 6.5 (61) <0.1 14037 20

[0278] The results are represented in Table 10 and in FIG. 36, Panels Aand B. These indicate that culture with anti-CD3/anti-CD28 antibodycoated beads resulted in decreased viral burden in all patient-derivedcells, including the cells cultured in the absence of antiretroviralagents. HIV-1 gag proviral DNA became undetectable in 6 of 7 culturesfrom patients that were cultured in the absence of antiretroviralagents, while HIV-1 gag RNA became undetectable in 5 of the 7 cultures.Culture supernatants were also sampled for p24 antigen at weekly tofortnightly intervals and antigen was not detected in 9 of the 10patients while in one patient (Patient 9, Table 10) decreasing levels ofp24 antigen with time were detected. Thus, expansion of T cells fromHIV-infected individuals does not require the presence of antiretroviraldrugs.

EXAMPLE 22 Anti-CD3/Anti-CD28 Mediated Expansion of Polyclonal CD4⁺ TCells from HIV-infected Individuals in Absence of Retroviral Treatment

[0279] This example shows that expansion of CD4⁺ T cells fromHIV-infected individual in the presence of anti-CD3/anti-CD28 antibodycoated beads is decreased in the presence of antiretroviral drugs.

[0280] CD4⁺ T cells from Patient 5 were cultured with anti-CD3/anti-CD28coated beads as described in Example 21, and further in the absence orin the presence of AZT (1 μM), DDI (5 μM) and Nevirapine (0.5 μM) andthe number of cells determined over a period of 50 days. The growthcurves are represented in FIG. 37, Panel A, and indicate that theantiretroviral drugs slow cell growth. Thus, expansion of T cells fromHIV-infected individuals using anti-CD3/anti-CD28 coated beads, which donot require the presence of antiretroviral drugs, allows for a morerapid expansion of the cell population than a culture in whichantiretrovial drugs are necessary.

[0281] Next, the antigen receptor diversity of the expanded populationsof CD4⁺ cells was assessed by flow cytometric measurement of the TCR VPrepertoire. CD4⁺ T cells were stained with monoclonal antibodies beforeculture (day 0) and after 27 days of culture. Reagents to identify Tcell receptor Vβ expression (T Cell Sciences, Woburn, Mass., andImmunotech, Westbrook Me.) and CD4 expression (Leu 3, Becton Dickinson)were used with flow cytometry. FIG. 37, Panel B, depicts the % cellsthat were positive for the various Vβ receptors. All patients haddiverse Vβ family expression similar to the input population of cells,consistent with a CD28-mediated polyclonal expansion of CD4⁺ lymphocytesfrom non HIV-infected individuals.

EXAMPLE 23 CD4⁺ T Cells from HIV-infected Individuals SecreteTh1-specific Cytokines Upon Stimulation with Anti-CD3/Anti-CD28 CoatedBeads

[0282] In this example, the types of cytokines produced by CD4⁺ T cellsfrom HIV-infected individuals cultured with anti-CD3/anti-CD28 coatedbeads were determined. Cultures from 8 patients in Table 10 were testedafter 10 to 20 days in culture: cells were collected, washed, and thelevel of cytokine secretion determined upon restimulation by placing thecells into fresh medium and restimulation with anti-CD3/anti-CD28 coatedbeads for 24 hours. Cell free supernatants were analyzed for IL-2, IFNγ,IL-4, IL-5, and TNF-α by ELISA using commercially available kits, asdescribed herein.

[0283]FIG. 38 represents the amount of cytokines produced. Consistentwith the amount of cytokines produced from non-HIV infected cellsstimulated with anti-CD3 and anti-CD28 antibodies, the cells from theHIV-infected patients secreted large amounts of Th1 cytokines afterstimulation with immobilized anti-CD3 plus anti-CD28 antibodies.

EXAMPLE 24 Large Scale Cultures of CD4⁺ T Cells from HIV-infectedIndividuals

[0284] The examples described above demonstrate the ability to growpolyclonal HIV-1-uninfected CD4⁺ T cells from HIV-infected donors, andthat the mechanism is independent of CD8⁺ T cells and antiretroviraldrugs. CD28 stimulation could provide a selective growth advantage tosubsets of cells that do not support infection. Alternatively, CD28stimulation could inhibit HIV-1 replication, selectively stimulate thedifferentiation of CD4 cells that do not support HIV-1 infection, orinduce death of the HIV-infected cells.

[0285] To determine whether enrichment for uninfected cells by limitingdilution through cells discarded during culture might account for theloss of HIV-1 infected cells, large-volume culture was performed byaddition of medium without removal of cells.

[0286] CD4⁺ T cells from Patient 5 were isolated, stimulated withanti-CD3/anti-CD28 coated beads and 4×10⁶ cells expanded to 2.7×10⁹ in a3-liter gas permeable culture bag. Culture for 25 days was performed byserial addition of fresh medium that did not contain antiretroviraldrugs and without discarding any cells. The cells were counted, mediumsampled for p24 and cells were analyzed for HIV-1 DNA by PCR at weeklyintervals. p24 remained undetectable throughout culture. Initial viralburden (HIV-l gag DNA) was 10⁷ copies/10⁵ cells, which decreased to 34copies/10⁵ cells during the initial 25 days of culture in the absence ofcell discards. Culture was then continued for a further 15 days and 1.7log 10 expansion in 25 cm² flasks using standard medium addition withcell discards in order to maintain a cell concentration of ˜1×10⁶/ml.During days 25 to 40, viral burden was further decreased from 34copies/10⁵ cells to undetectable levels (<5 copies/10⁵ cells).

[0287] These results indicate that CD28 stimulation permittedlarge-scale culture to >10¹⁰CD4⁺ T cells in the absence of cell discardsand antiretroviral drugs in patients with intermediate-stage HIV-1infection. These experiments also indicated that the frequency of HIV-1infected cells decreases during culture with anti-CD28 under theseconditions, and therefore, that this antiviral effect can not beattributed to serial replacement of medium and cells with fresh mediumduring the cell culture process.

EXAMPLE 25 Immobilized but not Soluble Anti-CD28 Antibody 9.3 PreventsInfection of CD4⁺ T Cells by HIV-1

[0288] The observed reduction in viral load during expansion of patientlymphocytes with immobilized anti-CD3/anti-CD28 antibodies is incontrast to previous studies showing that the addition of solubleanti-CD28 antibody to cultures of lymphocytes from HIV-1 infected donorsresulted in enhanced HIV-1 expression, and enhanced HIV-1 productionafter in vitro infection of CD4⁺ T cells (Asjo et al. J. Virol. 67, 4395(1993); Smithgall et al AIDS Res. Hum. Retroviruses 11, 885 (1995);Pinchuk et al. Immunity 1, 317 (1994)). To assess whether the mode ofCD28 stimulation might be important in determining the level of HIV-1replication after in vitro infection, CD8-depleted PBMC were cultured inthe presence of high-titer HIV-1_(Ba-L) and activated with anti-CD3antibody and either immobilized or soluble anti-CD28 antibody.

[0289] CD8-depleted PBMC were cultured for 2 days in PHA at 5 μg/ml andrIL-2 at 100 U/ml, anti-CD3 at 100 ng/ml and rIL-2 at 100 U/ml andsoluble anti-CD28 mAb 9.3 at 1 μg/ml, or anti-CD3/ anti-CD28 coatedbeads as described herein. HIV-1_(Ba-L) was added at day 0 at 2666TCID₅₀per 10⁶ cells. On day 2, virus was washed out of all cultures, cellswere replated in conditioned media obtained from companion cultures ofuninfected cells and harvested after a further 1 to 17 days of culturewith fresh media added as necessary to maintain the cells at aconcentration of 1-2×10⁶ cells per ml. Cell free supernatants wereharvested at 2 to 3 day intervals and p24 levels were determined byELISA.

[0290] The production of p24 is depicted in FIG. 39, Panel A, and thegrowth curve of the cells is depicted in FIG. 39, Panel B.PHA-stimulated cells and cells stimulated with soluble anti-CD3 andanti-CD28 developed high levels of p24 antigen. In marked contrast, thecultures stimulated with immobilized anti-CD3 and anti-CD28 did notcontain detectable levels of p24. The differences in HIV-1 p24 levelswere not due to differences in strength of T cell activation, as thegrowth of the cells during the experiment was equivalent with all formsof activation (FIG. 39, Panel B).

[0291] To further examine the effect of soluble versus immobilizedantibodies on the production of HIV by CD4⁺ T cells, CD8-depleted PBMCwere cultured for 2 days in soluble anti-CD3 OKT3 at 100 ng/ml andanti-CD28 9.3 antibody at 1 μg/ml, soluble anti-CD3 at 100 ng/ml andrIL-2 at 100 U/ml, soluble anti-CD3 at 100 ng/ml and soluble anti-CD28at 1 μg/ml and rIL-2 at 100 U/ml, soluble anti-CD3 at 100 ng/ml and CD28coated beads, or anti-CD3/anti-CD28 coated beads. HIV-1_(Ba-L) was addedon day 0 at 5000 TCID₅₀ per 106 cells. Cells were cultured for 2 days,washed to remove virus and supernatants harvested as described above.The results are presented in FIG. 39, Panel C. The results indicate thatthe lowest amounts of p24 antigen was produced when the cells werestimulated with immobilized anti-CD28 antibody.

[0292] Results similar to those presented in FIG. 39 were also observedwhen cell cultures containing purified CD4⁺ T cells were used ratherthan PBMC.

EXAMPLE 26 Inhibition of HIV Production by Immobilized Anti-CD28Antibody Acts Early in the HIV-1 Life Cycle

[0293] The above results indicate that depending on the mode of CD28receptor engagement, costimulation can enhance or potentially inhibitHIV-1 expression or the susceptibility to HIV-1 infection in CD4⁺ Tcells. To distinguish between these possibilities and ascertain thestage of this antiviral effect in the life cycle of HIV-1 infection,cells were stimulated with PHA or anti-CD28 for 3 days prior toinfection with high-titer HIV-I and quantitative PCR was used to assessthe kinetics of full-length gag DNA accumulation.

[0294] CD4⁺ T cells were cultured for 3 days with 5 μg/ml PHA +100 U/mlrIL-2, anti-CD3 OKT3 coated beads plus 100 U/ml rIL-2 oranti-CD3⁺CD28-coated beads as described in Example 25. The cells werecollected and incubated with DNAse-treated HIV-1_(Ba-L) at 7000TCID₅₀/1×10⁶ cells for 4 hours, washed 3 times (T=0), replated inconditioned medium obtained from companion cultures of uninfected cells,and harvested after a further 2 to 72 hours of culture. Frozen cellpellets containing 2×10⁶ cells were lysed and the amount of gag DNAdetermined by quantitative PCR, as described above.

[0295]FIG. 40, Panels A-D, represent the results. These indicate thatafter a four hour exposure to mV-1, cells previously stimulated witheither PHA plus IL-2 or immobilized anti-CD3 plus IL-2 had high levelsof viral gag DNA that was detected within 12 to 24 hours of culture. Incontrast, cells stimulated with immobilized anti-CD3 and anti-CD28 hadbackground or near-background levels of gag DNA at all time pointsassessed. Phosphorimage analysis indicated that there was >100-folddecrease in gag PCR product in CD3 plus CD28-stimulated cells incomparison to cells stimulated with PHA and IL-2.

[0296] Thus, stimulation of CD4⁺ T cells of HIV-infected individualswith anti-CD3/anti-CD28 coated beads inhibits production of viralparticles by acting at an early stage of the viral life cycle, prior tointegration. Furthermore, since CD4 receptor function remains normal incells stimulated with anti-CD3 and CD28, the induction of an HIV-1resistant state occurs downstream of HIV-1 binding.

EXAMPLE 27 CD28 Stimulation with Immobilized Anti-CD28 Antibody PreventsProduction of HIV Particles in a Dominant Manner

[0297] To determine whether immobilized anti-CD28 antibody stimulationcan prevent or reduce HIV infection of CD4⁺ T cells which are otherwisepermissive to HIV, CD4⁺ T cells (95% purity) were cultured for 3 days in5 g/ml PHA+100 U/ml IL-2, 5 μg/ml PHA+anti-CD28 coated beads oranti-CD3/anti-CD28 antibody coated beads as described in Example 26. Thecells were collected and infected with DNAse-treated HIV-1_(US1) isolate(Gartner et al. Science 233, 215 (1986); Mascola et al. J. Infect. Dis.169, 48 (1994)) at 1.5×10³TCID₅₀/1×10⁶ cells. Cells were collected aftera further 4 to 120 hours of culture for PCR analysis. HIV-1 gag andβ-Globin was quantitated from frozen cell pellets as described above.

[0298]FIG. 41 represents the amount of gag DNA in the cells followingculture for various times in the presence of PHA and IL-2, PHA andimmobilized anti-CD28 antibody, or anti-CD3 and anti-CD28 immobilizedantibodies. The amount of gag DNA is significantly reduced when thecells are cultured in the presence of immobilized anti-CD28 antibody.Thus, CD28 costimulation with immobilized anti-CD28 antibody confers amarked resistance to HIV-1 infection. The effect appeared to bedominant, as CD4⁺ cells cultured in conditions otherwise permissive toHIV-1 infection such as mitogenic lectins and IL-2 could not be infectedin the presence of immobilized CD28 costimulation. The protective effectof CD28 costimulation was prolonged, lasting for about 3 weeks ofculture. The effect did not appear to depend on the strain of virus usedfor infection, which is consistent with the ability of CD28costimulation to expand CD4⁺ cells from multiple patients infected withHIV-1.

[0299] Thus, the mode of costimulation permits the rescue of polyclonalCD4⁺ T cells from HIV-infected patients and regulates the susceptibilityof CD4⁺ T cells to HIV-1 infection. Cells stimulated with solubleanti-CD28 support high level HIV-1 infection, confirming previousstudies. In contrast, immobilized anti-CD28 had a potent inhibitoryeffect on HIV-1 replication, permitting the routine culture andexpansion of polyclonal CD4⁺ cells from HIV-1 infected patients in theabsence of antiretroviral agents. The mechanism is multifactorial andinvolves a prominent protective effect of CD28 stimulation against HIV-1infection as well as a proliferative advantage of HIV-uninfected CD4⁺cells over HIV-infected cells.

[0300] Furthermore, the effect of CD28 stimulation by, for example,immobilized anti-CD28 antibody may also provide protection againstinfection by other viruses, such as retroviruses, DNA viruses, and RNAviruses.

[0301] It has been previously reported that CD28 can stimulate distinctsignal transduction pathways, depending on the degree of CD28 receptoroligomerization (Nunes et al. J. Exp. Med. 180, 1067 (1994); Ledbetteret al. Blood 75, 1531 (1990)). It is possible that the permissive andinhibitory forms of CD28 costimulation reflect differential signaltransduction, and that distinct forms of signal transduction conferHIV-1 susceptible or resistant states. The CD28-mediated antiviraleffect described herein appears to be distinct from that previouslydescribed by Levy and others (Walker et al. Science 234, 1563 (1986)).

[0302] The culture of large numbers of CD4⁺ cells from HIV-infectedpatients has been difficult. Recent success at CD4⁺ cell expansion in asubset of HIV-infected patients required addition of multipleantiretroviral agents to cell culture medium to prevent viral expression(Haffar et al. PRoc. Natl. Acad. Sci. USA 90, 11094 (1993)). However,clinical utility was limited by drug-resistant virus breakthrough and arequirement for allogeneic feeder cells to restimulate lymphocytes.There are several therapeutic approaches for HIV-1 infection that mightbe facilitated by the present results. Ex vivo expansion of CD4⁺ T cellsmay permit immune reconstitution and vaccine therapies in patientsinvolving autologous transfusions of polyclonal or antigen-specific CD4⁺T cells. Moreover, autologous transfusions of CD4 lymphocytes mightprovide the immunologic help necessary to sustain CD8 T cell function.It is shown herein that while CD28 stimulation can prevent HIV-1infection and expression, it supports high transduction efficiencieswith Moloney leukemia-based retroviral vectors, so that culture systemsemploying CD28 costimulation might be useful to generate CD4⁺ cells forgene therapy as well as immunotherapy. Finally, our results indicatethat in vivo manipulation of CD28 interaction with B7 counter receptorshas the potential to enhance CD4 cell proliferation and prevent or limitHIV-1 viral spread in patients.

[0303] Equivalents

[0304] Those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, many equivalents to thespecific embodiments of the invention described herein. Such equivalentsare intended to be encompassed by the following claims.

0 SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES:14 (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 1491 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (iii)HYPOTHETICAL: no (iv) ANTI-SENSE: no (vi) ORIGINAL SOURCE: (A) ORGANISM:Homo sapien (F) TISSUE TYPE: lymphoid (G) CELL TYPE: B cell (H) CELLLINE: Raji (vii) IMMEDIATE SOURCE: (A) LIBRARY: cDNA in pCDM8 vector (B)CLONE: B7, Raji clone #13 (viii) POSITION IN GENOME: (A)CHROMOSOME/SEGMENT: 3 (ix) FEATURE: (A) NAME/KEY: Open reading frame(translated region) (B) LOCATION: 318 to 1181 bp (C) IDENTIFICATIONMETHOD: similarity to other pattern (ix) FEATURE: (A) NAME/KEY:Alternate polyadenylation signal (B) LOCATION: 1474 to 1479 bp (C)IDENTIFICATION METHOD: similarity to other pattern (x) PUBLICATIONINFORMATION: (A) AUTHORS: FREEMAN, GORDON J. FREEDMAN, ARNOLD S. SEGIL,JEFFREY M. LEE, GRACE WHITMAN, JAMES F. NADLER, LEE M. (B) TITLE: B7, ANew Member Of The Ig Superfamily With Unique Expression On Activated AndNeoplastic B Cells (C) JOURNAL: The Journal of Immunology (D) VOLUME:143 (E) ISSUE: 8 (F) PAGES: 2714-2722 (G) DATE: 15-OCT-1989 (H) RELEVANTRESIDUES IN SEQ ID NO:1: FROM 1 TO 1491 (xi) SEQUENCE DESCRIPTION: SEQID NO:1: CCAAAGAAAA AGTGATTTGT CATTGCTTTA TAGACTGTAA GAAGAGAACATCTCAGAAGT 60 GGAGTCTTAC CCTGAAATCA AAGGATTTAA AGAAAAAGTG GAATTTTTCTTCAGCAAGCT 120 GTGAAACTAA ATCCACAACC TTTGGAGACC CAGGAACACC CTCCAATCTCTGTGTGTTTT 180 GTAAACATCA CTGGAGGGTC TTCTACGTGA GCAATTGGAT TGTCATCAGCCCTGCCTGTT 240 TTGCACCTGG GAAGTGCCCT GGTCTTACTT GGGTCCAAAT TGTTGGCTTTCACTTTTGAC 300 CCTAAGCATC TGAAGCC ATG GGC CAC ACA CGG AGG CAG GGA ACATCA CCA TCC 353 Met Gly His Thr Arg Arg Gln Gly Thr Ser Pro Ser -30 -25AAG TGT CCA TAC CTG AAT TTC TTT CAG CTC TTG GTG CTG GCT GGT CTT 401 LysCys Pro Tyr Leu Asn Phe Phe Gln Leu Leu Val Leu Ala Gly Leu -20 -15 -10TCT CAC TTC TGT TCA GGT GTT ATC CAC GTG ACC AAG GAA GTG AAA GAA 449 SerHis Phe Cys Ser Gly Val Ile His Val Thr Lys Glu Val Lys Glu -5 1 5 10GTG GCA ACG CTG TCC TGT GGT CAC AAT GTT TCT GTT GAA GAG CTG GCA 497 ValAla Thr Leu Ser Cys Gly His Asn Val Ser Val Glu Glu Leu Ala 15 20 25 CAAACT CGC ATC TAC TGG CAA AAG GAG AAG AAA ATG GTG CTG ACT ATG 545 Gln ThrArg Ile Tyr Trp Gln Lys Glu Lys Lys Met Val Leu Thr Met 30 35 40 ATG TCTGGG GAC ATG AAT ATA TGG CCC GAG TAC AAG AAC CGG ACC ATC 593 Met Ser GlyAsp Met Asn Ile Trp Pro Glu Tyr Lys Asn Arg Thr Ile 45 50 55 TTT GAT ATCACT AAT AAC CTC TCC ATT GTG ATC CTG GCT CTG CGC CCA 641 Phe Asp Ile ThrAsn Asn Leu Ser Ile Val Ile Leu Ala Leu Arg Pro 60 65 70 TCT GAC GAG GGCACA TAC GAG TGT GTT GTT CTG AAG TAT GAA AAA GAC 689 Ser Asp Glu Gly ThrTyr Glu Cys Val Val Leu Lys Tyr Glu Lys Asp 75 80 85 90 GCT TTC AAG CGGGAA CAC CTG GCT GAA GTG ACG TTA TCA GTC AAA GCT 737 Ala Phe Lys Arg GluHis Leu Ala Glu Val Thr Leu Ser Val Lys Ala 95 100 105 GAC TTC CCT ACACCT AGT ATA TCT GAC TTT GAA ATT CCA ACT TCT AAT 785 Asp Phe Pro Thr ProSer Ile Ser Asp Phe Glu Ile Pro Thr Ser Asn 110 115 120 ATT AGA AGG ATAATT TGC TCA ACC TCT GGA GGT TTT CCA GAG CCT CAC 833 Ile Arg Arg Ile IleCys Ser Thr Ser Gly Gly Phe Pro Glu Pro His 125 130 135 CTC TCC TGG TTGGAA AAT GGA GAA GAA TTA AAT GCC ATC AAC ACA ACA 881 Leu Ser Trp Leu GluAsn Gly Glu Glu Leu Asn Ala Ile Asn Thr Thr 140 145 150 GTT TCC CAA GATCCT GAA ACT GAG CTC TAT GCT GTT AGC AGC AAA CTG 929 Val Ser Gln Asp ProGlu Thr Glu Leu Tyr Ala Val Ser Ser Lys Leu 155 160 165 170 GAT TTC AATATG ACA ACC AAC CAC AGC TTC ATG TGT CTC ATC AAG TAT 977 Asp Phe Asn MetThr Thr Asn His Ser Phe Met Cys Leu Ile Lys Tyr 175 180 185 GGA CAT TTAAGA GTG AAT CAG ACC TTC AAC TGG AAT ACA ACC AAG CAA 1025 Gly His Leu ArgVal Asn Gln Thr Phe Asn Trp Asn Thr Thr Lys Gln 190 195 200 GAG CAT TTTCCT GAT AAC CTG CTC CCA TCC TGG GCC ATT ACC TTA ATC 1073 Glu His Phe ProAsp Asn Leu Leu Pro Ser Trp Ala Ile Thr Leu Ile 205 210 215 TCA GTA AATGGA ATT TTT GTG ATA TGC TGC CTG ACC TAC TGC TTT GCC 1121 Ser Val Asn GlyIle Phe Val Ile Cys Cys Leu Thr Tyr Cys Phe Ala 220 225 230 CCA AGA TGCAGA GAG AGA AGG AGG AAT GAG AGA TTG AGA AGG GAA AGT 1169 Pro Arg Cys ArgGlu Arg Arg Arg Asn Glu Arg Leu Arg Arg Glu Ser 235 240 245 250 GTA CGCCCT GTA TAACAGTGTC CGCAGAAGCA AGGGGCTGAA AAGATCTGAA 1221 Val Arg Pro ValGGTAGCCTCC GTCATCTCTT CTGGGATACA TGGATCGTGG GGATCATGAG GCATTCTTCC 1281CTTAACAAAT TTAAGCTGTT TTACCCACTA CCTCACCTTC TTAAAAACCT CTTTCAGATT 1341AAGCTGAACA GTTACAAGAT GGCTGGCATC CCTCTCCTTT CTCCCCATAT GCAATTTGCT 1401TAATGTAACC TCTTCTTTTG CCATGTTTCC ATTCTGCCAT CTTGAATTGT CTTGTCAGCC 1461AATTCATTAT CTATTAAACA CTAATTTGAG 1491 (2) INFORMATION FOR SEQ ID NO:2:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 288 amino acids (B) TYPE:amino acid (C) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (A)DESCRIPTION: B cell activation antigen; natural ligand for CD28 T cellsurface antigen; transmembrane protein (ix) FEATURE: (A) NAME/KEY:signal sequence (B) LOCATION: -34 to -1 (C) IDENTIFICATION METHOD: aminoterminal sequencing of soluble protein (D) OTHER INFORMATION:hydrophobic (ix) FEATURE: (A) NAME/KEY: extracellular domain (B)LOCATION: 1 to 208 (C) IDENTIFICATION METHOD: similarity with knownsequence (ix) FEATURE: (A) NAME/KEY: transmembrane domain (B) LOCATION:209 to 235 (C) IDENTIFICATION METHOD: similarity with known sequence(ix) FEATURE: (A) NAME/KEY: intracellular domain (B) LOCATION: 236 to254 (C) IDENTIFICATION METHOD: similarity with known sequence (ix)FEATURE: (A) NAME/KEY: N-linked glycosylation (B) LOCATION: 19 to 21 (C)IDENTIFICATION METHOD: similarity with known sequence (ix) FEATURE: (A)NAME/KEY: N-linked glycosylation (B) LOCATION: 55 to 57 (C)IDENTIFICATION METHOD: similarity with known sequence (ix) FEATURE: (A)NAME/KEY: N-linked glycosylation (B) LOCATION: 64 to 66 (C)IDENTIFICATION METHOD: similarity with known sequence (ix) FEATURE: (A)NAME/KEY: N-linked glycosylation (B) LOCATION: 152 to 154 (C)IDENTIFICATION METHOD: similarity with known sequence (ix) FEATURE: (A)NAME/KEY: N-linked glycosylation (B) LOCATION: 173 to 175 (C)IDENTIFICATION METHOD: similarity with known sequence (ix) FEATURE: (A)NAME/KEY: N-linked glycosylation (B) LOCATION: 177 to 179 (C)IDENTIFICATION METHOD: similarity with known sequence (ix) FEATURE: (A)NAME/KEY: N-linked glycosylation (B) LOCATION: 192 to 194 (C)IDENTIFICATION METHOD: similarity with known sequence (ix) FEATURE: (A)NAME/KEY: N-linked glycosylation (B) LOCATION: 198 to 200 (C)IDENTIFICATION METHOD: similarity with known sequence (ix) FEATURE: (A)NAME/KEY: Ig V-set domain (B) LOCATION: 1 to 104 (C) IDENTIFICATIONMETHOD: similarity with known sequence (ix) FEATURE: (A) NAME/KEY: IgC-set domain (B) LOCATION: 105 to 202 (C) IDENTIFICATION METHOD:similarity with known sequence (x) PUBLICATION INFORMATION: (A) AUTHORS:FREEMAN, GORDON J. FREEDMAN, ARNOLD S. SEGIL, JEFFREY M. LEE, GRACEWHITMAN, JAMES F. NADLER, LEE M. (B) TITLE: B7, A New Member Of The IgSuperfamily With Unique Expression On Activated And Neoplastic B Cells(C) JOURNAL: The Journal of Immunology (D) VOLUME: 143 (E) ISSUE: 8 (F)PAGES: 2714-2722 (G) DATE: 15-OCT-1989 (H) RELEVANT RESIDUES IN SEQ IDNO:2: From -26 to 262 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met GlyHis Thr Arg Arg Gln Gly Thr Ser Pro Ser Lys Cys Pro Tyr -30 -25 -20 LeuAsn Phe Phe Gln Leu Leu Val Leu Ala Gly Leu Ser His Phe Cys -15 -10 -5Ser Gly Val Ile His Val Thr Lys Glu Val Lys Glu Val Ala Thr Leu -1 1 510 Ser Cys Gly His Asn Val Ser Val Glu Glu Leu Ala Gln Thr Arg Ile 15 2025 30 Tyr Trp Gln Lys Glu Lys Lys Met Val Leu Thr Met Met Ser Gly Asp 3540 45 Met Asn Ile Trp Pro Glu Tyr Lys Asn Arg Thr Ile Phe Asp Ile Thr 5055 60 Asn Asn Leu Ser Ile Val Ile Leu Ala Leu Arg Pro Ser Asp Glu Gly 6570 75 Thr Tyr Glu Cys Val Val Leu Lys Tyr Glu Lys Asp Ala Phe Lys Arg 8085 90 Glu His Leu Ala Glu Val Thr Leu Ser Val Lys Ala Asp Phe Pro Thr 95100 105 110 Pro Ser Ile Ser Asp Phe Glu Ile Pro Thr Ser Asn Ile Arg ArgIle 115 120 125 Ile Cys Ser Thr Ser Gly Gly Phe Pro Glu Pro His Leu SerTrp Leu 130 135 140 Glu Asn Gly Glu Glu Leu Asn Ala Ile Asn Thr Thr ValSer Gln Asp 145 150 155 Pro Glu Thr Glu Leu Tyr Ala Val Ser Ser Lys LeuAsp Phe Asn Met 160 165 170 Thr Thr Asn His Ser Phe Met Cys Leu Ile LysTyr Gly His Leu Arg 175 180 185 190 Val Asn Gln Thr Phe Asn Trp Asn ThrThr Lys Gln Glu His Phe Pro 195 200 205 Asp Asn Leu Leu Pro Ser Trp AlaIle Thr Leu Ile Ser Val Asn Gly 210 215 220 Ile Phe Val Ile Cys Cys LeuThr Tyr Cys Phe Ala Pro Arg Cys Arg 225 230 235 Glu Arg Arg Arg Asn GluArg Leu Arg Arg Glu Ser Val Arg Pro Val 240 245 250 (2) INFORMATION FORSEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1120 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: cDNA (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION:107..1093 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: CACAGGGTGA AAGCTTTGCTTCTCTGCTGC TGTAACAGGG ACTAGCACAG ACACACGGAT 60 GAGTGGGGTC ATTTCCAGATATTAGGTCAC AGCAGAAGCA GCCAAA ATG GAT CCC 115 Met Asp Pro 1 CAG TGC ACTATG GGA CTG AGT AAC ATT CTC TTT GTG ATG GCC TTC CTG 163 Gln Cys Thr MetGly Leu Ser Asn Ile Leu Phe Val Met Ala Phe Leu 5 10 15 CTC TCT GGT GCTGCT CCT CTG AAG ATT CAA GCT TAT TTC AAT GAG ACT 211 Leu Ser Gly Ala AlaPro Leu Lys Ile Gln Ala Tyr Phe Asn Glu Thr 20 25 30 35 GCA GAC CTG CCATGC CAA TTT GCA AAC TCT CAA AAC CAA AGC CTG AGT 259 Ala Asp Leu Pro CysGln Phe Ala Asn Ser Gln Asn Gln Ser Leu Ser 40 45 50 GAG CTA GTA GTA TTTTGG CAG GAC CAG GAA AAC TTG GTT CTG AAT GAG 307 Glu Leu Val Val Phe TrpGln Asp Gln Glu Asn Leu Val Leu Asn Glu 55 60 65 GTA TAC TTA GGC AAA GAGAAA TTT GAC AGT GTT CAT TCC AAG TAT ATG 355 Val Tyr Leu Gly Lys Glu LysPhe Asp Ser Val His Ser Lys Tyr Met 70 75 80 GGC CGC ACA AGT TTT GAT TCGGAC AGT TGG ACC CTG AGA CTT CAC AAT 403 Gly Arg Thr Ser Phe Asp Ser AspSer Trp Thr Leu Arg Leu His Asn 85 90 95 CTT CAG ATC AAG GAC AAG GGC TTGTAT CAA TGT ATC ATC CAT CAC AAA 451 Leu Gln Ile Lys Asp Lys Gly Leu TyrGln Cys Ile Ile His His Lys 100 105 110 115 AAG CCC ACA GGA ATG ATT CGCATC CAC CAG ATG AAT TCT GAA CTG TCA 499 Lys Pro Thr Gly Met Ile Arg IleHis Gln Met Asn Ser Glu Leu Ser 120 125 130 GTG CTT GCT AAC TTC AGT CAACCT GAA ATA GTA CCA ATT TCT AAT ATA 547 Val Leu Ala Asn Phe Ser Gln ProGlu Ile Val Pro Ile Ser Asn Ile 135 140 145 ACA GAA AAT GTG TAC ATA AATTTG ACC TGC TCA TCT ATA CAC GGT TAC 595 Thr Glu Asn Val Tyr Ile Asn LeuThr Cys Ser Ser Ile His Gly Tyr 150 155 160 CCA GAA CCT AAG AAG ATG AGTGTT TTG CTA AGA ACC AAG AAT TCA ACT 643 Pro Glu Pro Lys Lys Met Ser ValLeu Leu Arg Thr Lys Asn Ser Thr 165 170 175 ATC GAG TAT GAT GGT ATT ATGCAG AAA TCT CAA GAT AAT GTC ACA GAA 691 Ile Glu Tyr Asp Gly Ile Met GlnLys Ser Gln Asp Asn Val Thr Glu 180 185 190 195 CTG TAC GAC GTT TCC ATCAGC TTG TCT GTT TCA TTC CCT GAT GTT ACG 739 Leu Tyr Asp Val Ser Ile SerLeu Ser Val Ser Phe Pro Asp Val Thr 200 205 210 AGC AAT ATG ACC ATC TTCTGT ATT CTG GAA ACT GAC AAG ACG CGG CTT 787 Ser Asn Met Thr Ile Phe CysIle Leu Glu Thr Asp Lys Thr Arg Leu 215 220 225 TTA TCT TCA CCT TTC TCTATA GAG CTT GAG GAC CCT CAG CCT CCC CCA 835 Leu Ser Ser Pro Phe Ser IleGlu Leu Glu Asp Pro Gln Pro Pro Pro 230 235 240 GAC CAC ATT CCT TGG ATTACA GCT GTA CTT CCA ACA GTT ATT ATA TGT 883 Asp His Ile Pro Trp Ile ThrAla Val Leu Pro Thr Val Ile Ile Cys 245 250 255 GTG ATG GTT TTC TGT CTAATT CTA TGG AAA TGG AAG AAG AAG AAG CGG 931 Val Met Val Phe Cys Leu IleLeu Trp Lys Trp Lys Lys Lys Lys Arg 260 265 270 275 CCT CGC AAC TCT TATAAA TGT GGA ACC AAC ACA ATG GAG AGG GAA GAG 979 Pro Arg Asn Ser Tyr LysCys Gly Thr Asn Thr Met Glu Arg Glu Glu 280 285 290 AGT GAA CAG ACC AAGAAA AGA GAA AAA ATC CAT ATA CCT GAA AGA TCT 1027 Ser Glu Gln Thr Lys LysArg Glu Lys Ile His Ile Pro Glu Arg Ser 295 300 305 GAT GAA GCC CAG CGTGTT TTT AAA AGT TCG AAG ACA TCT TCA TGC GAC 1075 Asp Glu Ala Gln Arg ValPhe Lys Ser Ser Lys Thr Ser Ser Cys Asp 310 315 320 AAA AGT GAT ACA TGTTTT TAATTAAAGA GTAAAGCCCA AAAAAAA 1120 Lys Ser Asp Thr Cys Phe 325 (2)INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:329 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULETYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Met Asp Pro GlnCys Thr Met Gly Leu Ser Asn Ile Leu Phe Val Met 1 5 10 15 Ala Phe LeuLeu Ser Gly Ala Ala Pro Leu Lys Ile Gln Ala Tyr Phe 20 25 30 Asn Glu ThrAla Asp Leu Pro Cys Gln Phe Ala Asn Ser Gln Asn Gln 35 40 45 Ser Leu SerGlu Leu Val Val Phe Trp Gln Asp Gln Glu Asn Leu Val 50 55 60 Leu Asn GluVal Tyr Leu Gly Lys Glu Lys Phe Asp Ser Val His Ser 65 70 75 80 Lys TyrMet Gly Arg Thr Ser Phe Asp Ser Asp Ser Trp Thr Leu Arg 85 90 95 Leu HisAsn Leu Gln Ile Lys Asp Lys Gly Leu Tyr Gln Cys Ile Ile 100 105 110 HisHis Lys Lys Pro Thr Gly Met Ile Arg Ile His Gln Met Asn Ser 115 120 125Glu Leu Ser Val Leu Ala Asn Phe Ser Gln Pro Glu Ile Val Pro Ile 130 135140 Ser Asn Ile Thr Glu Asn Val Tyr Ile Asn Leu Thr Cys Ser Ser Ile 145150 155 160 His Gly Tyr Pro Glu Pro Lys Lys Met Ser Val Leu Leu Arg ThrLys 165 170 175 Asn Ser Thr Ile Glu Tyr Asp Gly Ile Met Gln Lys Ser GlnAsp Asn 180 185 190 Val Thr Glu Leu Tyr Asp Val Ser Ile Ser Leu Ser ValSer Phe Pro 195 200 205 Asp Val Thr Ser Asn Met Thr Ile Phe Cys Ile LeuGlu Thr Asp Lys 210 215 220 Thr Arg Leu Leu Ser Ser Pro Phe Ser Ile GluLeu Glu Asp Pro Gln 225 230 235 240 Pro Pro Pro Asp His Ile Pro Trp IleThr Ala Val Leu Pro Thr Val 245 250 255 Ile Ile Cys Val Met Val Phe CysLeu Ile Leu Trp Lys Trp Lys Lys 260 265 270 Lys Lys Arg Pro Arg Asn SerTyr Lys Cys Gly Thr Asn Thr Met Glu 275 280 285 Arg Glu Glu Ser Glu GlnThr Lys Lys Arg Glu Lys Ile His Ile Pro 290 295 300 Glu Arg Ser Asp GluAla Gln Arg Val Phe Lys Ser Ser Lys Thr Ser 305 310 315 320 Ser Cys AspLys Ser Asp Thr Cys Phe 325 (2) INFORMATION FOR SEQ ID NO:5: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acid(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: (A)NAME/KEY: misc_feature (B) LOCATION: 1 (D) OTHER INFORMATION: /label=Xaais any amino acid (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION:3 (D) OTHER INFORMATION: /label=Xaa is any amino acid (ix) FEATURE: (A)NAME/KEY: misc_feature (B) LOCATION: 6 (D) OTHER INFORMATION: /label=Xaais any amino acid (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION:7 (D) OTHER INFORMATION: /label=Xaa is any amino acid (ix) FEATURE: (A)NAME/KEY: misc_feature (B) LOCATION: 8 (D) OTHER INFORMATION: /label=Xaais Asp or Glu (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION: 9(D) OTHER INFORMATION: /label=Xaa is any amino acid (xi) SEQUENCEDESCRIPTION: SEQ ID NO:5: Xaa Gly Xaa Trp Leu Xaa Xaa Xaa Xaa 5 (2)INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:227 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULETYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Pro Val Lys GlyGly Thr Lys Cys Ile Lys Tyr Leu Leu Phe Gly Phe 5 10 15 Asn Phe Ile PheTrp Leu Ala Gly Ile Ala Val Leu Ala Ile Gly Leu 20 25 30 Trp Leu Arg PheAsp Ser Gln Thr Lys Ser Ile Phe Glu Gln Glu Thr 35 40 45 Asn Asn Asn AsnSer Ser Phe Tyr Thr Gly Val Tyr Ile Leu Ile Gly 50 55 60 Ala Gly Ala LeuMet Met Leu Val Gly Phe Leu Gly Cys Cys Gly Ala 65 70 75 80 Val Gln GluSer Gln Cys Met Leu Gly Leu Phe Phe Gly Phe Leu Leu 85 90 95 Val Ile PheAla Ile Glu Ile Ala Ala Ala Ile Trp Gly Tyr Ser His 100 105 110 Lys AspGlu Val Ile Lys Glu Val Gln Glu Phe Tyr Lys Asp Thr Tyr 115 120 125 AsnLys Leu Lys Thr Lys Asp Glu Pro Gln Arg Glu Thr Leu Lys Ala 130 135 140Ile His Tyr Ala Leu Asn Cys Cys Gly Leu Ala Gly Gly Val Glu Gln 145 150155 160 Phe Ile Ser Asp Ile Cys Pro Lys Lys Asp Val Leu Glu Thr Phe Thr165 170 175 Val Lys Ser Cys Pro Asp Ala Ile Lys Glu Val Phe Asp Asn LysPhe 180 185 190 His Ile Ile Gly Ala Val Gly Ile Gly Ile Ala Val Val MetIle Phe 195 200 205 Gly Met Ile Phe Ser Met Ile Leu Cys Cys Ala Ile ArgArg Asn Arg 210 215 220 Glu Met Val 225 (2) INFORMATION FOR SEQ ID NO:7:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 7 amino acids (B) TYPE: aminoacid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCEDESCRIPTION: SEQ ID NO:7: Gly Leu Trp Leu Arg Phe Asp 1 5 (2)INFORMATION FOR SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:20 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULETYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: His Gln Phe CysAsp His Trp Gly Cys Trp Leu Leu Arg Glu Thr His 1 5 10 15 Ile Phe ThrPro 20 (2) INFORMATION FOR SEQ ID NO:9: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: LeuArg Leu Val Leu Glu Asp Pro Gly Ile Trp Leu Arg Pro Asp Tyr 1 5 10 15Phe Phe Pro Ala 20 (2) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 7 amino acids (B) TYPE: amino acid (D)TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION:SEQ ID NO:10: Gly Cys Trp Leu Leu Arg Glu 1 5 (2) INFORMATION FOR SEQ IDNO:11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 7 amino acids (B) TYPE:amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi)SEQUENCE DESCRIPTION: SEQ ID NO:11: Gly Ile Trp Leu Arg Pro Asp 1 5 (2)INFORMATION FOR SEQ ID NO:12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:6 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULETYPE: peptide (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION: 2(D) OTHER INFORMATION: /label=Xaa is any amino acid (ix) FEATURE: (A)NAME/KEY: misc_feature (B) LOCATION: 5 (D) OTHER INFORMATION: /label=Xaais any amino acid (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION:6 (D) OTHER INFORMATION: /label=Xaa is Asp or Glu (xi) SEQUENCEDESCRIPTION: SEQ ID NO:12: Gly Xaa Trp Leu Xaa Xaa 1 5 (2) INFORMATIONFOR SEQ ID NO:13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 basepairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:CTTTAGAGCA CA 12 (2) INFORMATION FOR SEQ ID NO:14: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 8 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi)SEQUENCE DESCRIPTION: SEQ ID NO:14: CTCTAAAG 8

1. A method for inducing a population of T cells to proliferate,comprising: a) activating a population of T cells; b) stimulating anaccessory molecule on the surface of the T cells with a ligand whichbinds the accessory molecule, the activating and stimulating stepsthereby inducing proliferation of the T cells; c) monitoringproliferation of the T cells in response to continuing exposure to theligand; and d) reactivating and restimulating the T cells when the rateof T cell proliferation has decreased to induce further proliferation ofthe T cells.